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4,547 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What happens with the viral seeding at the local endothelium? | {
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4,548 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | How long does the process of giving rise to primary viremia for hantavirus infections? | {
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4,549 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What happens by the time that secondary viremia emerges? | {
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" the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass"
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4,550 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | How is the the expression of proinflammatory cytokines induced? | {
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4,551 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | For HCPS, what does that expression favor? | {
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4,552 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | For HCPS, what does that expression spare? | {
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4,553 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What happens for HFRS? | {
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4,554 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What is considered to be a requirement for the development of systemic disease symptoms? | {
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4,555 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What do those abnormalities sometimes culminate in? | {
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4,556 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What leads to death in most fatal cases of HCPS? | {
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4,557 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What leads to death in most fatal cases of HCPS? | {
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4,558 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What potential mechanism, could be presumed to underlie the pathogenesis of HCPS? | {
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" Innate immune mechanisms."
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4,559 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What potential mechanism, could be presumed to underlie the pathogenesis of HCPS? | {
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4,560 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What potential mechanism, could be presumed to underlie the pathogenesis of HCPS? | {
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4,561 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What explanation have some investigators favored for much of the capillary leak? | {
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4,562 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What animal models exist for both the asymptomatic carriage of PUUV and SNV? | {
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4,563 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | For what can the Syrian hamster model used? | {
"answer_start": [
20140
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"text": [
"ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed"
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4,564 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What does the hamster model for HCPS caused by? | {
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4,565 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | Typically how long do the hamsters die post-inoculation? | {
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4,566 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What does the microscopic examination of the lung reveal, as with human HCPS? | {
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4,567 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What do ANDV-infected hamsters fitted with physiologic monitoring devices exhibit? | {
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4,568 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | What do diminished pulse pressures, tachycardia, and hypotension in ANDV infected hamsters appear to closely mimic? | {
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"the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS "
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4,570 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | Compared to humans, what do ANDV infected hamsters exhibit? | {
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"exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes"
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4,572 | Hantaviruses in the Americas and Their Role as Emerging Pathogens
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/
SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101
Authors: Hjelle, Brian; Torres-Pérez, Fernando
Date: 2010-11-25
DOI: 10.3390/v2122559
License: cc-by
Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.
Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] .
Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus.
During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] .
A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] .
Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic.
While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] .
The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖).
Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] .
Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] .
By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma.
The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases.
Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include:
(1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] .
(2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] .
(3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections.
Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] .
The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] .
Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] .
The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] .
A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis.
Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] .
While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] .
Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil).
Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] .
The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] .
Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements.
Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] .
Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] .
The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] .
Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] .
Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] .
Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses.
In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease. | With what have three studies correlated plasma viral RNA? | {
"answer_start": [
15442
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"text": [
"with disease severity for HCPS and HFRS,"
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809 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What are examples of delivery vectors for commercial anti-Salmonella vaccines? | {
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"Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals"
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798 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What can be a factor in using common vectors for the delivery of vaccines? | {
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800 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | Is a pre-existing immune response to commonly used delivery vector an advantage or a disadvantage? | {
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802 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What bacterial delivery vectors have been tested in animal hosts? | {
"answer_start": [
1614
],
"text": [
" attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts"
]
} | false |
804 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | Which bacteial delivery vectors have gained favor for vaccines? | {
"answer_start": [
2079
],
"text": [
"Bacteria such as E. coli and lactic acid bacteria "
]
} | false |
805 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | Why are E Coli and lactic acid are safe choices as delivery vectors for vaccines? | {
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812 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is Listeria? | {
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813 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is the advantage of Listeria as a delivery vector for vaccines? | {
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"The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells "
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817 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What are examples of viral vectors for delivering vaccines? | {
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"recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] "
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825 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | Which viral vaccine delivery vector was first licensed? | {
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"YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2"
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831 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What are examples of attenuated poxvirus vaccine delivery vectors? | {
"answer_start": [
6675
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"text": [
"modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains"
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872 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is the connection between chicken and Salmonella? | {
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17653
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"text": [
"Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. "
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833 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | Why are some poxvirus ideally suited as vaccine delivery vectors? | {
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836 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is the advantage of adenovirus as vaccine delivery vector? | {
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"adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses "
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838 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What are important criteria for selecting vaccine delivery vectors? | {
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841 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What are important criteria for selecting vaccine delivery vectors? | {
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845 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What are important criteria for selecting vaccine delivery vectors? | {
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858 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What happens when a recipient of a vaccine has immune response to the delivery vector? | {
"answer_start": [
9357
],
"text": [
"considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response)"
]
} | false |
864 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is the effect of host immune response to the delivery vector on the efficacy of vaccination? | {
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9980
],
"text": [
"for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen."
]
} | false |
861 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is the effect of host immune response to the delivery vector on the efficacy of vaccination? | {
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870 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is an example of the effect of immunity to the delivery vector on the efficacy of vaccination? | {
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"mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses."
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874 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is the effect of host immune response to viral delivery vectors in the efficacy of vaccination? | {
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"pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] "
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875 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is the effect of host immune response to the viral delivery vector on the efficacy of vaccination? | {
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876 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What is the effect of host immune response to the delivery vector on the efficacy of vaccination? | {
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877 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What are methods to avoid the effect vector immunity on the efficacy of vaccination? | {
"answer_start": [
23489
],
"text": [
" the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) "
]
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878 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | What are methods to avoid the effect of vector immune response on the efficacy of vaccination? | {
"answer_start": [
23859
],
"text": [
"The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .\n"
]
} | false |
879 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | How does cell-mediated immunity to viral delivery vector, reduce the immune response to vaccine? | {
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" this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes"
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880 | Pre-existing immunity against vaccine vectors – friend or foe?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/
SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c
Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M.
Date: 2013-01-27
DOI: 10.1099/mic.0.049601-0
License: cc-by
Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses.
Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) .
Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) .
However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) .
As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen.
In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector.
Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 .
The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) .
A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 .
Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic
Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated.
Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced.
It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) .
Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1.
Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed.
Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations.
Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 .
There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) .
As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe.
Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) .
Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) .
All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) .
For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.
In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry.
To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity. | How can vectors for which host has immunity, be used differently to increase the efficacy of vaccination? | {
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" it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context.\n\nIn addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response."
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601 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | How does the PED virus transmit between animals? | {
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602 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | How can Bacilius subtilis be used as an oral vaccine? | {
"answer_start": [
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603 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What cells are infected by the PED virus? | {
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604 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What kind of immune responses are most effective in preventing PED virus? | {
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605 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What intestinal factors may reduce the effectiveness of orally-administered immunizations? | {
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3759
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606 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What is Bacillus subtilis? | {
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607 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What is the role of dendritic cells in the immune response? | {
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608 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | Where do dendritic cells exist in the body? | {
"answer_start": [
4991
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"text": [
"gut-associated lymphoid tissue (GALT)"
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609 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What are the components of the gut-associated lymphoid tissue? | {
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610 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What type of cells form the intestinal mucosal barrier? | {
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611 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What factors determine an effective mucosal immune response? | {
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612 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What is an effective indicator of a vaccine's ability to generate an immune response? | {
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613 | Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/
SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8
Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian
Date: 2019-03-15
DOI: 10.1042/bsr20182028
License: cc-by
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets.
Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] .
As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] .
It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics.
As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system.
In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection.
The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China.
Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days.
Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde.
The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] .
The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] .
The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] .
Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately.
PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant.
PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine.
Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system.
The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01).
CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets.
SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection.
A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets.
Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection.
The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection.
Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] .
It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells.
Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection.
As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10.
In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets. | What is interleukin-1Beta? | {
"answer_start": [
22214
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"pro-inflammatory cytokines"
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1,197 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | When was the first case of COVID-19 identified? | {
"answer_start": [
981
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"text": [
"Wuhan City, China"
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1,199 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | Where did SARS-CoV-2 originate? | {
"answer_start": [
981
],
"text": [
"Wuhan City, China"
]
} | false |
1,201 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | In what year did the first SARS epidemic occur? | {
"answer_start": [
5102
],
"text": [
" rapid and transparent sharing of information between countries and agencies"
]
} | false |
1,202 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | When was World Health Organization (WHO) first notified about the SARS-CoV-2 epidemic in Wuhan City, China? | {
"answer_start": [
1268
],
"text": [
"31 December"
]
} | false |
1,203 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | When did we discover that SARS-CoV-2, which causes COVID-19, was a novel coronavirus? | {
"answer_start": [
1304
],
"text": [
"26 January 2020"
]
} | false |
1,204 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | How long did it take to identify the cause of COVID-19? | {
"answer_start": [
1292
],
"text": [
"4 weeks"
]
} | false |
1,205 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | What type of test was initially developed to screen for SARS-CoV-2? | {
"answer_start": [
1445
],
"text": [
" reverse transcription polymerase chain reaction"
]
} | false |
1,206 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | How big was the temporary hospital built in Wuhan City for treatment of COVID-19 patients? | {
"answer_start": [
1743
],
"text": [
"1000 bed hospital"
]
} | false |
1,207 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | How long did it take China to build the temporary hospital in Wuhan for COVID-19 patients? | {
"answer_start": [
1773
],
"text": [
"10 days"
]
} | false |
1,208 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | What is a key factor in managing emerging infectious disease threats? | {
"answer_start": [
5113
],
"text": [
"transparent sharing of information between countries and agencies"
]
} | false |
1,209 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | In what year did the MERS epidemic occur? | {
"answer_start": [
5511
],
"text": [
"2012"
]
} | false |
1,210 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | How long did it take to publish the full genomic sequence of SARS-CoV-2 after it was identified? | {
"answer_start": [
5951
],
"text": [
"2 weeks"
]
} | false |
1,211 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | What was the fatality rate for SARS-CoV? | {
"answer_start": [
7310
],
"text": [
"10%"
]
} | false |
1,212 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | What was the fatality rate for MERS? | {
"answer_start": [
7331
],
"text": [
"34%"
]
} | false |
1,213 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | What are some challenges associated with using media and social media to capture information about an emerging epidemic? | {
"answer_start": [
8273
],
"text": [
"the volume and diversity of the information available and the relative lack of verification mechanisms"
]
} | false |
1,214 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | What are the risks of health workers failing to wash hands? | {
"answer_start": [
8828
],
"text": [
"autoinfection, but also in infection of patients hospitalised for other causes when they provide care"
]
} | false |
1,215 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | Who is at risk when health workers fail to wash their hands? | {
"answer_start": [
8967
],
"text": [
" the health worker, but also for their families and the communities in which they live"
]
} | false |
1,216 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | What was the R0 of SARS in absence of control measures? | {
"answer_start": [
9947
],
"text": [
"3"
]
} | false |
1,217 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | What is superspreading? | {
"answer_start": [
9409
],
"text": [
"where a case infected significantly more contacts than the average"
]
} | false |
1,218 | SARS to novel coronavirus – old lessons and new lessons
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026896/
SHA: 5d254ed178c092d3639ce70ae9653593acc471f9
Authors: McCloskey, Brian; Heymann, David L.
Date: 2020-02-05
DOI: 10.1017/s0950268820000254
License: cc-by
Abstract: The response to the novel coronavirus outbreak in China suggests that many of the lessons from the 2003 SARS epidemic have been implemented and the response improved as a consequence. Nevertheless some questions remain and not all lessons have been successful. The national and international response demonstrates the complex link between public health, science and politics when an outbreak threatens to impact on global economies and reputations. The unprecedented measures implemented in China are a bold attempt to control the outbreak – we need to understand their effectiveness to balance costs and benefits for similar events in the future.
Text: On 29 December 2019 clinicians in a hospital in Wuhan City, China noticed a clustering of cases of unusual pneumonia (with the first case identified at that time on 12 December) with an apparent link to a market that sells live fish, poultry and animals to the public. This event was reported to the World Health Organisation (WHO) on 31 December [1]. Within 4 weeks, by 26 January 2020, the causative organism had been identified as a novel coronavirus, the genome of the virus had been sequenced and published, reverse transcription polymerase chain reaction tests had been developed, the WHO R&D Blueprint had been activated to accelerate diagnostics, therapeutics and vaccine development and a candidate vaccine was ready for initial laboratory testing. Currently Chinese health authorities are building a 1000 bed hospital in Wuhan in 10 days.
By 26 January also, almost 50 million people in Wuhan and neighbouring cities had effectively been placed in quarantine while the WHO had determined that the event should not yet be declared as a Public Health Emergency of International Concern (PHEIC) [2] and had recommended no specific travel restrictions. The WHO have emphasised the importance of exit screening at ports in countries showing transmission of the novel coronavirus and have provided guidance for countries implementing entry screening at airports while acknowledging that evidence for the effectiveness of entry screening is equivocal.
This response is one of the swiftest, coordinated global responses to an emerging infectious disease the world has seen in modern times, but is it the appropriate response, will it be effective and is it sustainable?
According to the situation report published by the WHO on 28 January 2020 [3], a total of 2798 confirmed 2019-nCoV cases have been reported globally; of these, 2761 cases were from China, including Hong Kong (8 cases), Macau (5) and Taipei (4). Thirty-seven confirmed cases have been reported outside of China in eleven countries in Europe, North America, Australia and Asia; of these 37 exported cases, 36 had a travel history from China or an epidemiological link to a case from China. Of the confirmed cases in China, 461 have been reported as severely ill, with 80 deaths to date.
This outbreak and the response to it illustrate some key issues about how global preparedness and response capacity for outbreaks have evolved over almost two decades since the severe acute respiratory syndrome (SARS) epidemic of 2002/3 and what lessons have, or have not, been learned. It also raises questions about the impact these lessons have had on the way agencies and governments respond to these events and about the role of the WHO and the International Health Regulations (IHR).
One of the critical lessons from the SARS experience was the absolute necessity to be able to coordinate the international resources that are available in an outbreak and to get them focussed on identifying priorities and solving problems. The WHO established the means to do this for SARS and it has since been further developed and integrated into global preparedness, especially after the West Africa Ebola epidemic. Organisations such as the Global Outbreak Alert and Response Network (GOARN), the Coalition for Epidemic Preparedness Innovations (CEPI), the Global Research Collaboration For Infectious Disease Preparedness (GloPID-R) and the Global Initiative on Sharing All Influenza Data (GISAID) have been supported by the WHO Research Blueprint and its Global Coordinating Mechanism to provide a forum where those with the expertise and capacity to contribute to managing new threats can come together both between and during outbreaks to develop innovative solutions to emerging problems. This global coordination has been active in the novel coronavirus outbreak. WHO's response system includes three virtual groups based on those developed for SARS to collate real time information to inform real time guidelines, and a first candidate vaccine is ready for laboratory testing within 4 weeks of the virus being identified.
Another key factor in successfully preventing and managing emerging threats is the rapid and transparent sharing of information between countries and agencies. There was extensive criticism of China for its perceived failure to share information about the emerging SARS infection early enough in the outbreak to allow countries to prepare and respond. There were similar concerns about information sharing as Middle East Respiratory Syndrome (MERS) emerged and evolved in the Middle East in 2012, particularly in Saudi Arabia, and about the emergence of Ebola in West Africa in 2014.
On this occasion information sharing seems to have been rapid and effective (while recognising that the information available in the early stages of an outbreak is always less than the global community would like). The WHO was notified of the original clustering within days and the full genomic sequence of the new virus was published less than 2 weeks after the cluster was first detected. The WHO has expressed its satisfaction with the actions of the Chinese authorities in sharing information with the WHO.
Working with journalists and the media to help them understand the science and epidemiology, particularly in a fast moving event, will improve risk communication to the public and reduce inappropriate concerns and panic.
While reporting of this outbreak shows signs of the efforts of epidemiologists, infectious disease experts, national and international public health agencies and others engaging with journalists, there are also signs that this is not yet achieving it's goal. For example, the public perception is that the increase in case numbers reported daily by the Chinese authorities represents a daily escalation in the epidemic while the reality is that these numbers are also the result of active, aggressive, case finding in China and some of these cases are 'old' cases newly recognised as being due to the novel coronavirus. Similarly the virus is usually described by the media as 'deadly' and although this is true in the sense that it has caused deaths, the nuances of uncertain case fatality rates in the early stages of an outbreak are not being communicated. The current estimated case fatality rate seems to be around 3% which is significant but not comparable to the 10% rate for SARS or 34% reported for MERS. These misperceptions are still driving public anxiety.
To supplement formal reporting mechanisms between countries and with WHO (including the IHR), the use of informal mechanisms such as media and social media reports was advocated in the light of the SARS experience. There are now globally several systems that provide collated information from informal reporting including networks of experts and scanning of media and social media. These contribute to, and amplify, epidemic intelligence and are being integrated with national and international surveillance systems.
The value, and the challenges, of this additional source of information has been evident in the current outbreak. The value comes from ensuring that early indications of cases beyond the initial outbreak city have been detected and can supplement the global risk assessment and monitoring of the evolution of the outbreak. The challenges lie in the volume and diversity of the information available and the relative lack of verification mechanisms, such that one of these systems (ProMed) has commented that it was becoming increasingly difficult to assimilate the information being supplied [4] and to make meaningful interpretations.
Early in the outbreak it was reported that health workers had not been infected. This was reassuring because it is health workers who many times, and inadvertently, amplify transmission. Failure to wash hands between patients, for example, can result not only in autoinfection, but also in infection of patients hospitalised for other causes when they provide care. Autoinfection is not only a risk for the health worker, but also for their families and the communities in which they live, depending on the transmissibility and means of transmission. More recently infection, and at least one death, in health workers has been confirmed. Although not unexpected this does add to the epidemiological risk.
A characteristic of the SARS outbreak was the variability of transmissibility between cases and the occurrence of 'superspreading events' where a case infected significantly more contacts than the average. This was also seen with MERS in the outbreak in the Republic of Korea (RoK). In this current novel coronavirus outbreak, such superspreading events have not been documented but the epidemiology is still not clear. Confirming whether or not this is happening must be an urgent task for the Chinese investigation. Modellers have suggested reproductive rates (R 0 ) of 3.8 (95% confidence interval, 3.6-4.0) [5] and 2.6 (1.5-3.5) [6] ; R 0 for SARS was estimated at around 3 in the absence of control measures [7] .
The economic impact of major outbreaks can be substantial for the affected country. This was seen clearly in SARS, MERS in RoK and Ebola in West Africa. One analyst estimates that the current coronavirus outbreak's likely impact will range from a 0.8% cut to real GDP if the epidemic is controlled within 3 months, to a 1.9% cost to GDP if the epidemic lasts 9 months [8] . This may increase substantially in the light of the extended restrictions on movement, and therefore trade and commerce, within China.
The emergence of a significant respiratory illness linked to a novel coronavirus represents a test of the global capacity to detect and mange emerging disease threats. Its emergence in China adds an additional dimension in the light of previous experience with SARS. The timing of the outbreak immediately before the Chinese Lunar New Year with its attendant population movements adds extra risk and urgency to the response.
The rapid sharing of information in this outbreak and the speed of the coordinated response both in the country and internationally suggest that lessons have been learned from SARS that improve global capacity. The international networks and forums that now exist have facilitated the bringing together of expertise from around the world to focus research and development efforts and maximise the impact.
At this early stage in the outbreak information remains incomplete and key clinical and epidemiological questions have not yet been answered, but the deficit seems to be due more to the constraints of investigating an emerging disease than to any unwillingness to engage and share information with partners.
There are some indications of areas where further improvement is necessary. The global media response to the unfolding events has been relatively balanced and informed but the nuances of the evolving situation have not been critically examined in partnership with the media and as a result the public perception of the risk may be exaggeratedalthough it of course remains possible that the outbreak will develop in a way that matches up to the perceived risk. The lack of appreciation of the uncertainties in determining a meaningful case fatality rate and the significance of ascertainment bias at the beginning of an outbreak, along with the impact of aggressive case finding on case numbers, are examples of where understanding could be improved. This is always a challenging process when balancing the resources focussed on analysing the situation on the ground with resources directed at interpreting the information for journalists but in SARS, the R 0 was seen to decrease in response to information reaching the public and the public then adopting risk reduction actions [6] ; so accurate public risk communication is critical to success. It would be helpful to find a forum where this can be explored with the media community after the event.
The increase in access to early information from diverse sources including media and social media adds an important dimension to identifying and tracking new events globally and is a key part of the overall epidemic intelligence system. However, it is also a potential source of disinformation. When, as has been seen in this outbreak, the volume of information coming in exceeds any capacity to collate and analyse it and to attempt to cross-reference and verify separate items, there is a risk that the information fuels speculation and media and public concern. Again there is a fine balance between information that encourages appropriate risk avoidance actions and information that encourages inappropriate actions; however the public health is usually better served by more information rather than less.
The role of a declaration of a PHEIC in managing a serious outbreak has been questioned in the light of Ebola in West Africa and in the Democratic Republic of Congo [9] and has been challenged again with this outbreak. The binary nature of a PHEIC declaration (either an event is a PHEIC or it isn'tthere are no intermediate options) and the specificity of the three defined criteria for a PHEIC have caused difficulty for Emergency Committees in considering whether a given event should be a PHEIC. The lack of a clear understanding of what a PHEIC declaration is meant to achieve adds to the Emergency Committee's difficulties, as does the relative paucity of clinical and epidemiological answers at this stage of the investigation. In this instance the Emergency Committee were divided in coming to a conclusion but decided on balance that the current situation, although an emergency, should not as yet be declared a PHEIC [2]. As with Ebola in the DRC, there has been criticism of the WHO for this decision but, as with Ebola, it is not immediately clear what would be different in the response if a PHEIC was declared.
The WHO is working on improving the way in which Emergency Committees develop their advice for the Director General but, as recommended by this Emergency Committee and the post-Ebola IHR Review Committee in 2015, the development of an intermediate alert alongside WHO's risk assessment process may be helpful.
A key function of a PHEIC declaration is that it is the (only) gateway to the WHO Temporary Recommendations on possible travel and trade restrictions to limit international spread of a disease. In this case several countries globally had already implemented entry screening at airports and China had begun closing down international travel from Wuhan before the Emergency Committee had finished their deliberations. While the WHO would not, and could not, interfere with the sovereign decisions of member states, the lack of influence on travel and trade decisions could prove problematic.
Alongside the speed of the response in this outbreak, we have seen dramatic changes in the scale of the response. The imposition of very extensive quarantine measures on millions of people as an attempt to break the transmission of the virus is unprecedented. We do not know whether they will be effective; indeed we do not know how we will determine if they have been effectivewhat end point can we measure that will provide an answer to that question? If recent suggestions that people infected with this coronavirus may be infectious while incubating or asymptomatic, and the reports that up to 5 m people left Wuhan before the travel restrictions were imposed, are confirmed, the efficacy of these control measures will be more challenged.
Given the likely impact on at least the Chinese economy and probably the global economy, it will be important to understand the role and the effectiveness of public health measures on this scale for the future.
However, the imposition of these dramatic measures does also raise a wider question: if there is an impact from these measures, what other countries would (or could) implement such measures? Would other countries accept the self-imposed economic damage that China has accepted to try and contain this outbreak? Is it reasonable to consider that national governments would close down public transport into and out of London, New York or Paris in the week before Christmas even if it were shown to be an effective control measure?
These decisions and questions cross the interface between public health, science and politics. The response to this outbreak in
China was inevitably influenced by the historical reaction to the country's response to SARS and the world's suspicion of China's lack of cooperation at that time. The current response is therefore framed within a context of not wanting to be seen to be behaving in the same way with this event.
This may indicate another impact of the SARS (and MERS and Ebola) experience on the response to subsequent outbreaksa tendency to look at worst case scenarios and respond accordingly and a fear of 'getting it wrong'. This can deter leaders at all levels, from outbreak teams to national governments, from making judgements when all the information they would like is not available in case those judgments turn out to be wrong when the full information becomes available.
In emergency response it is generally better to over-react and then scale back if necessary rather than under-react and then act too late. Response should be on a 'no regrets' basismake the best decisions possible on the basis of the best information and science available at the time but do not judge or criticise if later information suggests a different course of action. The early response must recognise what is known and what is not known and look at what of the unknowns can reasonably be estimated by reference to previous outbreaks, similar pathogens, early reporting and modelling, etc. The risk assessment and response can then be modified and refined as information on the unknowns evolves.
Key to that approach, however, is confidence that decisions will not be criticised based on information that was not available at the time. It is also important to be ready to change decisions when the available information changessomething that both scientists and politicians can find difficult.
In that context, China should not be judged for implementing what might appear to be extreme measures but China should also be prepared to discontinue the measures quickly if evidence suggests they are not the best way to solve the problem. By closing airports the international spread from Wuhan may be decreased, but success will depend on how effective the measures really are at stopping people moving out of the affected area as well as on the behaviour of the virus. As always, only time will tellbut time is scarce. | How many people may have left Wuhan before travel restrictions were imposed? | {
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"5 m people"
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1,939 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | How many severe cases of influenza-related illnesses are reported per year? | {
"answer_start": [
1629
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"text": [
"Between 3-5 million"
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1,940 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | How many influenza-related deaths are reported each year? | {
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1695
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"over 250 000"
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1,941 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | What is the mortality rate of the H5N1 strain of influenza? | {
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1,942 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | What cells are the main target of the influenza A virus in the lungs? | {
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"primary human alveolar epithelial type II (ATII) cells"
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1,944 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | How many extracellular domains are in the CEAMCAM1 protein? | {
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1,945 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | Where is CEACAM1 expressed in the body? | {
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1,946 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | What motifs are absent in the short form of CEACAM1 protein? | {
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1,947 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | What are the most common isoforms of CEACAM1? | {
"answer_start": [
6858
],
"text": [
"CEACAM1-4L and CEACAM1-3L"
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} | false |
1,948 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | How do CEACAM1 and CEACAM5 interact? | {
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1,949 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | What are the SRC-family of kinases? | {
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1,950 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | What triggers the release of pro-inflammatory cytokines/chemokines to assist in viral clearance? | {
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1,951 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | What mediates the anti-apoptosis of neutrophils? | {
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"Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3"
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1,952 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | How do natural killer cells fight influenza viruses? | {
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1,953 | Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195505/
SHA: ef58c6e981a08c85d2c0efb80e5b32b075f660b4
Authors: Ye, Siying; Cowled, Christopher J.; Yap, Cheng-Hon; Stambas, John
Date: 2018-10-19
DOI: 10.1038/s41598-018-33605-6
License: cc-by
Abstract: Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.
Text: Influenza viruses cause acute and highly contagious seasonal respiratory disease in all age groups. Between 3-5 million cases of severe influenza-related illness and over 250 000 deaths are reported every year. In addition to constant seasonal outbreaks, highly pathogenic avian influenza (HPAI) strains, such as H5N1, remain an ongoing pandemic threat with recent WHO figures showing 454 confirmed laboratory infections and a mortality rate of 53%. It is important to note that humans have very little pre-existing immunity towards avian influenza virus strains. Moreover, there is no commercially available human H5N1 vaccine. Given the potential for H5N1 viruses to trigger a pandemic 1,2 , there is an urgent need to develop novel therapeutic interventions to combat known deficiencies in our ability to control outbreaks. Current seasonal influenza virus prophylactic and therapeutic strategies involve the use of vaccination and antivirals. Vaccine efficacy is highly variable as evidenced by a particularly severe 2017/18 epidemic, and frequent re-formulation of the vaccine is required to combat ongoing mutations in the influenza virus genome. In addition, antiviral resistance has been reported for many circulating strains, including the avian influenza H7N9 virus that emerged in 2013 3, 4 . Influenza A viruses have also been shown to target and hijack multiple host cellular pathways to promote survival and replication 5, 6 . As such, there is increasing evidence to suggest that targeting host pathways will influence virus replication, inflammation, immunity and pathology 5, 7 . Alternative intervention strategies based on modulation of the host response could be used to supplement the current prophylactic and therapeutic protocols.
While the impact of influenza virus infection has been relatively well studied in animal models 8, 9 , human cellular responses are poorly defined due to the lack of available human autopsy material, especially from HPAI virus-infected patients. In the present study, we characterized influenza virus infection of primary human alveolar epithelial type II (ATII) cells isolated from normal human lung tissue donated by patients undergoing lung resection. ATII cells are a physiologically relevant infection model as they are a main target for influenza A viruses when entering the respiratory tract 10 . Human host gene expression following HPAI H5N1 virus (A/Chicken/ Vietnam/0008/04) infection of primary ATII cells was analyzed using Illumina HiSeq deep sequencing. In order to gain a better understanding of the mechanisms underlying modulation of host immunity in an anti-inflammatory environment, we also analyzed changes in gene expression following HPAI H5N1 infection in the presence of the reactive oxygen species (ROS) inhibitor, apocynin, a compound known to interfere with NADPH oxidase subunit assembly 5, 6 .
The HiSeq analysis described herein has focused on differentially regulated genes following H5N1 infection. Several criteria were considered when choosing a "hit" for further study. These included: (1) Novelty; has this gene been studied before in the context of influenza virus infection/pathogenesis? (2) Immunoregulation; does this gene have a regulatory role in host immune responses so that it has the potential to be manipulated to improve immunity? (3) Therapeutic reagents; are there any existing commercially available therapeutic reagents, such as specific inhibitors or inhibitory antibodies that can be utilized for in vitro and in vivo study in order to optimize therapeutic strategies? (4) Animal models; is there a knock-out mouse model available for in vivo influenza infection studies? Based on these criteria, carcinoembryonic-antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) was chosen as a key gene of interest. CEACAM1 (also known as BGP or CD66) is expressed on epithelial and endothelial cells 11 , as well as B cells, T cells, neutrophils, NK cells, macrophages and dendritic cells (DCs) [12] [13] [14] . Human CEACAM1 has been shown to act as a receptor for several human bacterial and fungal pathogens, including Haemophilus influenza, Escherichia coli, Salmonella typhi and Candida albicans, but has not as yet been implicated in virus entry [15] [16] [17] . There is however emerging evidence to suggest that CEACAM1 is involved in host immunity as enhanced expression in lymphocytes was detected in pregnant women infected with cytomegalovirus 18 and in cervical tissue isolated from patients with papillomavirus infection 19 .
Eleven CEACAM1 splice variants have been reported in humans 20 . CEACAM1 isoforms (Uniprot P13688-1 to -11) can differ in the number of immunoglobulin-like domains present, in the presence or absence of a transmembrane domain and/or the length of their cytoplasmic tail (i.e. L, long or S, short). The full-length human CEACAM1 protein (CEACAM1-4L) consists of four extracellular domains (one extracellular immunoglobulin variable-region-like (IgV-like) domain and three immunoglobulin constant region 2-like (IgC2-like) domains), a transmembrane domain, and a long (L) cytoplasmic tail. The long cytoplasmic tail contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are absent in the short form 20 . The most common isoforms expressed by human immune cells are CEACAM1-4L and CEACAM1-3L 21 . CEACAM1 interacts homophilically with itself 22 or heterophilically with CEACAM5 (a related CEACAM family member) 23 . The dimeric state allows recruitment of signaling molecules such as SRC-family kinases, including the tyrosine phosphatase SRC homology 2 (SH2)-domain containing protein tyrosine phosphatase 1 (SHP1) and SHP2 members to phosphorylate ITIMs 24 . As such, the presence or absence of ITIMs in CEACAM1 isoforms influences signaling properties and downstream cellular function. CEACAM1 homophilic or heterophilic interactions and ITIM phosphorylation are critical for many biological processes, including regulation of lymphocyte function, immunosurveillance, cell growth and differentiation 25, 26 and neutrophil activation and adhesion to target cells during inflammatory responses 27 . It should be noted that CEACAM1 expression has been modulated in vivo using an anti-CEACAM1 antibody (MRG1) to inhibit CEACAM1-positive melanoma xenograft growth in SCID/NOD mice 28 . MRG1 blocked CEACAM1 homophilic interactions that inhibit T cell effector function, enhancing the killing of CEACAM1+ melanoma cells by T cells 28 . This highlights a potential intervention pathway that can be exploited in other disease processes, including virus infection. In addition, Ceacam1-knockout mice are available for further in vivo infection studies.
Our results show that CEACAM1 mRNA and protein expression levels were highly elevated following HPAI H5N1 infection. Furthermore, small interfering RNA (siRNA)-mediated inhibition of CEACAM1 reduced inflammatory cytokine and chemokine production, and more importantly, inhibited H5N1 virus replication in primary human ATII cells and in the continuous human type II respiratory epithelial A549 cell line. Taken together, these observations suggest that CEACAM1 is an attractive candidate for modulating influenza-specific immunity. In summary, our study has identified a novel target that may influence HPAI H5N1 immunity and serves to highlight the importance of manipulating host responses as a way of improving disease outcomes in the context of virus infection.
Three experimental groups were included in the HiSeq analysis of H5N1 infection in the presence or absence of the ROS inhibitor, apocynin: (i) uninfected cells treated with 1% DMSO (vehicle control) (ND), (ii) H5N1-infected cells treated with 1% DMSO (HD) and (iii) H5N1-infected cells treated with 1 mM apocynin dissolved in DMSO (HA). These three groups were assessed using pairwise comparisons: ND vs. HD, ND vs. HA, and HD vs. HA. H5N1 infection and apocynin treatment induce differential expression of host genes. ATII cells isolated from human patients 29, 30 were infected with H5N1 on the apical side at a multiplicity of infection (MOI) of 2 for 24 hours and RNA extracted. HiSeq was performed on samples and reads mapped to the human genome where they were then assembled into transcriptomes for differential expression analysis. A total of 13,649 genes were identified with FPKM (fragments per kilobase of exon per million fragments mapped) > 1 in at least one of the three experimental groups. A total of 623 genes were significantly upregulated and 239 genes were significantly downregulated (q value < 0.05, ≥2-fold change) following H5N1 infection (ND vs. HD) ( Fig. 1A ; Table S1 ). HPAI H5N1 infection of ATII cells activated an antiviral state as evidenced by the upregulation of numerous interferon-induced genes, genes associated with pathogen defense, cell proliferation, apoptosis, and metabolism (Table 1; Table S2 ). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping showed that many of the upregulated genes in the HD group were mapped to TNF signaling (hsa04668), Toll-like receptor signaling (hsa04620), cytokine-cytokine receptor interaction (hsa04060) and RIG-I-like receptor signaling (hsa04622) ( In the H5N1-infected and apocynin-treated (HA) group, a large number of genes were also significantly upregulated (509 genes) or downregulated (782 genes) ( Fig. 1B ; Table S1 ) relative to the ND control group. Whilst a subset of genes was differentially expressed in both the HD and HA groups, either being upregulated (247 genes, Fig. 1D ) or downregulated (146 genes, Fig. 1E ), a majority of genes did not in fact overlap between the HD and HA groups (Fig. 1D , E). This suggests that apocynin treatment can affect gene expression independent of H5N1 infection. Gene Ontology (GO) enrichment analysis of genes upregulated by apocynin showed the involvement of the type I interferon signaling pathway (GO:0060337), the defense response to virus (GO:0009615), negative regulation of viral processes (GO:48525) and the response to stress (GO:0006950) ( Table S2 , "ND vs. HA Up"). Genes downregulated by apocynin include those that are involved in cell adhesion (GO:0007155), regulation of cell migration (GO:0030334), regulation of cell proliferation (GO:0042127), signal transduction (GO:0007165) and oxidation-reduction processes (GO:0055114) ( Table S2 , "ND vs. HA Down").
A total of 623 genes were upregulated following H5N1 infection ("ND vs. HD Up", Fig. 1F ). By overlapping the two lists of genes from "ND vs. HD Up" and "HD vs. HA Down", 245 genes were shown to be downregulated in the presence of apocynin (Fig. 1F ). By overlapping three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Up", 55 genes out of the 245 genes (190 plus 55 genes) were present in all three lists (Fig. 1G) , indicating that these 55 genes were significantly inhibited by apocynin but to a level that was still significantly higher than that in uninfected cells. The 55 genes include those involved in influenza A immunity (hsa05164; DDX58, IFIH1, IFNB1, MYD88, PML, STAT2), Jak-STAT signaling (hsa04630; IFNB1, IL15RA, IL22RA1, STAT2), RIG-I-like receptor signaling (hsa04622; DDX58, IFIH1, IFNB1) and Antigen processing and presentation (hsa04612; TAP2, TAP1, HLA-DOB) (Tables S3 and S4) . Therefore, critical immune responses induced following H5N1 infection were not dampened following apocynin treatment. The remaining 190 of 245 genes were not present in the "ND vs. HA Up" list, suggesting that those genes were significantly inhibited by apocynin to a level that was similar to uninfected control cells (Fig. 1G ). The 190 genes include those involved in TNF signaling (hsa04668; CASP10, CCL2, CCL5, CFLAR, CXCL5, END1, IL6, TRAF1, VEGFC), cytokine-cytokine receptor interaction (hsa04060; VEGFC, IL6, CCL2, CXCL5, CXCL16, IL2RG, CD40, CCL5, CCL7, IL1A), NF-kappa B signaling pathway (hsa04064: TRAF1, CFLAR, CARD11, TNFSF13B, TICAM1, CD40) and PI3K-Akt signaling (hsa04151; CCND1, GNB4, IL2RG, IL6, ITGA2, JAK2, LAMA1, MYC, IPK3AP1, TLR2, VEGFC) (Tables S3 and S4 ). This is consistent with the role of apocynin in reducing inflammation 31 . By overlapping the three lists of genes from "ND vs. HD Up", "HD vs. HA Down" and "ND vs. HA Down", 11 genes were found in all three comparisons (Fig. 1H ). This suggests that these 11 genes are upregulated following H5N1 infection and are significantly reduced by apocynin treatment to a level lower than that observed in uninfected control cells (Fig. 1H ). Among these were inflammatory cytokines/chemokines genes, including CXCL5, IL1A, AXL (a member of the TAM receptor family of receptor tyrosine kinases) and TMEM173/STING (Stimulator of IFN Genes) (Table S4) .
Our previous study demonstrated that H5N1 infection of A549 cells in the presence of apocynin enhanced expression of negative regulators of cytokine signaling (SOCS), SOCS1 and SOCS3 6 . This, in turn, resulted in a reduction of H5N1-stimulated cytokine and chemokine production (IL6, IFNB1, CXCL10 and CCL5 in A549 cells), which was not attributed to lower virus replication as virus titers were not affected by apocynin treatment 6 . We performed a qRT-PCR analysis on the same RNA samples submitted for HiSeq analysis to validate HiSeq results. IL6 ( Fig. 2A) , IFNB1 (Fig. 2B) , CXCL10 (Fig. 2C ), and CCL5 ( Fig. 2D ) gene expression was significantly elevated in ATII cells following infection and was reduced by the addition of apocynin (except for IFNB1). Consistent with previous findings in A549 cells 6 , H5N1 infection alone induced the expression of SOCS1 as shown by HiSeq and qRT-PCR analysis (Fig. 2E ). Apocynin treatment further increased SOCS1 mRNA expression (Fig. 2E ). Although HiSeq analysis did not detect a statistically significant increase of SOCS1 following apocynin treatment, the Log2 fold-changes in SOCS1 gene expression were similar between the HD and HA groups (4.8-fold vs 4.0-fold) (Fig. 2E ). HiSeq analysis of SOCS3 transcription showed significant increase following H5N1 infection and apocynin treatment (Fig. 2F ). qRT-PCR analysis showed that although SOCS3 mRNA was only slightly increased following H5N1 infection, it was further significantly upregulated in the presence Table 2 . Representatives of over-represented KEGG pathways with a maximum P-value of 0.05 and the number of genes contributing to each pathway that is significantly upregulated following H5N1 infection ("ND vs. HD Up"). The full list of KEGG pathways is presented in Table S3 . of apocynin (Fig. 2F) . Therefore, apocynin also contributes to the reduction of H5N1-stimulated cytokine and chemokine production in ATII cells. Apocynin, a compound that inhibits production of ROS, has been shown to influence influenza-specific responses in vitro 6 and in vivo 5 . Although virus titers are not affected by apocynin treatment in vitro 6 , some anti-viral activity is observed in vivo when mice have been infected with a low pathogenic A/HongKong/X31 H3N2 virus 6 . HiSeq analysis of HPAI H5N1 virus gene transcription showed that although there was a trend for increased influenza virus gene expression following apocynin treatment, only influenza non-structural (NS) gene expression was significantly increased (Fig. 2G) . The reduced cytokine and chemokine production in H5N1-infected ATII cells ( Fig. 2A-F) is unlikely to be associated with lower virus replication.
GO enrichment analysis was performed on genes that were significantly upregulated following HPAI H5N1 infection in ATII cells in the presence or absence of apocynin to identify over-presented GO terms. Many of the H5N1-upregulated genes were broadly involved in defense response (GO:0006952), response to external biotic stimulus (GO:0043207), immune system processes (GO:0002376), cytokine-mediated signaling pathway (GO:0019221) and type I interferon signaling pathway (GO:0060337) ( Table 1; Table S2 ). In addition, many of the H5N1-upregulated genes mapped to metabolic pathways (hsa01100), cytokine-cytokine receptor interaction (hsa04060), Influenza A (hsa05164), TNF signaling (hsa04668) or Jak-STAT signaling (hsa04630) (Table S3) . However, not all the H5N1-upregulated genes in these pathways were inhibited by apocynin treatment as mentioned above ( Fig. 1F ; Table S3 ). . Fold-changes following qRT-PCR analysis were calculated using 2 −ΔΔCt method (right Y axis) normalized to β-actin and compared with the ND group. Data from HiSeq was calculated as Log2 fold-change (left Y axis) compared with the ND group. IFNB1 transcription was not detected in ND, therefore HiSeq IFNB1 data from HD and HA groups was expressed as FPKM. *p < 0.05 and **p < 0.01, ***p < 0.001 compared with ND; # p < 0.05, ## p < 0.01, compared with HD. (G) Hiseq analysis of H5N1 influenza virus gene expression profiles with or without apocynin treatment in primary human ATII cells. # p < 0.05, compared with HD. Upregulation of the cell adhesion molecule CEACAM1 in H5N1-infected ATII cells. The cell adhesion molecule CEACAM1 has been shown to be critical for the regulation of immune responses during infection, inflammation and cancer 20 . The CEACAM1 transcript was significantly upregulated following H5N1 infection (Fig. 3A) . In contrast, a related member of the CEACAM family, CEACAM5, was not affected by H5N1 infection (Fig. 3B) . It is also worth noting that more reads were obtained for CEACAM5 (>1000 FPKM) (Fig. 3B ) than CEACAM1 (~7 FPKM) (Fig. 3A) in uninfected ATII cells, which is consistent with their normal expression patterns in human lung tissue 32 . Therefore, although CEACAM1 forms heterodimers with CEACAM5 23 , the higher basal expression of CEACAM5 in ATII cells may explain why its expression was not enhanced by H5N1 infection. Endogenous CEACAM1 protein expression was also analyzed in uninfected or influenza virus-infected A549 (Fig. 3C ) and ATII cells (Fig. 3D ). CEACAM1 protein expression was slightly, but not significantly, increased in A549 cells infected with A/Puerto Rico/8/1934 H1N1 (PR8) virus for 24 or 48 hours when compared to uninfected cells (Fig. 3C ). No significant difference in CEACAM1 protein levels were observed at various MOIs (2, 5 or 10) or between the 24 and 48 hpi timepoints (Fig. 3C) .
After examing CEACAM1 protein expression following infection with PR8 virus in A549 cells, CEACAM1 protein expression was then examined in primary human ATII cells infected with HPAI H5N1 and compared to PR8 virus infection (Fig. 3D) . ATII cells were infected with PR8 virus at a MOI of 2, a dose that induced upregulation of cytokines and influenza Matrix (M) gene analyzed by qRT-PCR (data not shown). Lower MOIs of 0.5, 1 and 2 of HPAI H5N1 were tested due to the strong cytopathogenic effect H5N1 causes at higher MOIs. Endogenous CEACAM1 protein levels were significantly and similarly elevated in H5N1-infected ATII cells at the three MOIs tested. CEACAM1 protein expression in ATII cells infected with H5N1 at MOIs of 0.5 were higher at 48 hpi than those observed at 24 hpi (Fig. 3D ). HPAI H5N1 virus infection at MOIs of 0.5, 1 and 2 stimulated higher endogenous levels of CEACAM1 protein expression when compared to PR8 virus infection at a MOI of 2 at the corresponding time point (a maximum ~9-fold increase induced by H5N1 at MOIs of 0.5 and 1 at 48 hpi when compared to PR8 at MOI of 2), suggesting a possible role for CEACAM1 in influenza virus pathogenicity (Fig. 3D ).
In order to understand the role of CEACAM1 in influenza pathogenesis, A549 and ATII cells were transfected with siCEACAM1 to knockdown endogenous CEACAM1 protein expression. ATII and A549 cells were transfected with siCEACAM1 or siNeg negative control. The expression of four main CEACAM1 variants, CEACAM1-4L, -4S, -3L and -3S, and CEACAM1 protein were analyzed using SYBR Green qRT-PCR and Western blotting, respectively. SYBR Green qRT-PCR analysis showed that ATII cells transfected with 15 pmol of siCEACAM1 significantly reduced the expression of CEACAM1-4L and -4S when compared to siNeg control, while the expression of CEACAM1-3L and -3S was not altered (Fig. 4A ). CEACAM1 protein expression was reduced by approximately 50% in both ATII and A549 cells following siCEACAM1 transfection when compared with siNeg-transfected cells (Fig. 4B) . Increasing doses of siCEACAM1 (10, 15 and 20 pmol) did not further downregulate CEACAM1 protein expression in A549 cells (Fig. 4B ). As such, 15 pmol of siCEACAM1 was chosen for subsequent knockdown studies in both ATII and A549 cells. It is important to note that the anti-CEACAM1 antibody only detects L isoforms based on epitope information provided by Abcam. Therefore, observed reductions in CEACAM1 protein expression can be attributed mainly to the abolishment of CEACAM1-4L.
The functional consequences of CEACAM1 knockdown were then examined in ATII and A549 cells following H5N1 infection. IL6, IFNB1, CXCL10, CCL5 and TNF production was analyzed in H5N1-infected ATII and A549 cells using qRT-PCR. ATII (Fig. 5A ) and A549 cells (Fig. 5B) transfected with siCEACAM1 showed significantly lower expression of IL6, CXCL10 and CCL5 when compared with siNeg-transfected cells. However, the expression of the anti-viral cytokine, IFNB1, was not affected in both cells types. In addition, TNF expression, which can be induced by type I IFNs 33 , was significantly lower in siCEACAM1-transfected A549 cells (Fig. 5B) , but was not affected in siCEACAM1-transfected ATII cells (Fig. 5A) . Hypercytokinemia or "cytokine storm" in H5N1 and H7N9 virus-infected patients is thought to contribute to inflammatory tissue damage 34, 35 . Downregulation of CEACAM1 in the context of severe viral infection may reduce inflammation caused by H5N1 infection without dampening the antiviral response. Furthermore, virus replication was significantly reduced by 5.2-fold in ATII (Figs. 5C) and 4.8-fold in A549 cells (Fig. 5D ) transfected with siCEACAM1 when compared with siNeg-transfected cells. Virus titers in siNeg-transfected control cells were not significantly different from those observed in mock-transfected control cells (Fig. 5C,D) .
Influenza viruses utilize host cellular machinery to manipulate normal cell processes in order to promote replication and evade host immune responses. Studies in the field are increasingly focused on understanding and modifying key host factors in order to ameliorate disease. Examples include modulation of ROS to reduce inflammation 5 and inhibition of NFκB and mitogenic Raf/MEK/ERK kinase cascade activation to suppress viral replication 36, 37 . These host targeting strategies will offer an alternative to current interventions that are focused on targeting the virus. In the present study, we analyzed human host gene expression profiles following HPAI H5N1 infection and treatment with the antioxidant, apocynin. As expected, genes that were significantly upregulated following H5N1 infection were involved in biological processes, including cytokine signaling, immunity and apoptosis. In addition, H5N1-upregulated genes were also involved in regulation of protein phosphorylation, cellular metabolism and cell proliferation, which are thought to be exploited by viruses for replication 38 . Apocynin treatment had both anti-viral (Tables S2-S4) 5 and pro-viral impact (Fig. 2G) , which is not surprising as ROS are potent microbicidal agents, as well as important immune signaling molecules at different concentrations 39 . In our hands, apocynin treatment reduced H5N1-induced inflammation, but also impacted the cellular defense response, cytokine production and cytokine-mediated signaling. Importantly, critical antiviral responses were not compromised, i.e. expression of pattern recognition receptors (e.g. DDX58 (RIG-I), TLRs, IFIH1 (MDA5)) was not downregulated (Table S1 ). Given the significant interference of influenza viruses on host immunity, we focused our attention on key regulators of the immune response. Through HiSeq analysis, we identified the cell adhesion molecule CEACAM1 as a critical regulator of immunity. Knockdown of endogenous CEACAM1 inhibited H5N1 virus replication and reduced H5N1-stimulated inflammatory cytokine/chemokine production. H5N1 infection resulted in significant upregulation of a number of inflammatory cytokines/chemokines genes, including AXL and STING, which were significantly reduced by apocynin treatment to a level lower than that observed in uninfected cells (Table S4) . It has been previously demonstrated that anti-AXL antibody treatment of PR8-infected mice significantly reduced lung inflammation and virus titers 40 . STING has been shown to be important for promoting anti-viral responses, as STING-knockout THP-1 cells produce less type I IFN following influenza A virus infection 41 . Reduction of STING gene expression or other anti-viral factors (e.g. IFNB1, MX1, ISG15; Table S1 ) by apocynin, may in part, explain the slight increase in influenza gene transcription following apocynin treatment (Fig. 2G) . These results also suggest that apocynin treatment may reduce H5N1-induced inflammation and apoptosis. Indeed, the anti-inflammatory and anti-apoptotic effects of apocynin have been shown previously in a number of disease models, including diabetes mellitus 42 , myocardial infarction 43 , neuroinflammation 44 and influenza virus infection 6 .
Recognition of intracellular viral RNA by pattern recognition receptors (PRRs) triggers the release of pro-inflammatory cytokines/chemokines that recruit innate immune cells, such as neutrophils and NK cells, to the site of infection to assist in viral clearance 45 . Neutrophils exert their cytotoxic function by first attaching to influenza-infected epithelial cells via adhesion molecules, such as CEACAM1 46 . Moreover, studies have indicated that influenza virus infection promotes neutrophil apoptosis 47 , delaying virus elimination 48 . Phosphorylation of CEACAM1 ITIM motifs and activation of caspase-3 is critical for mediating anti-apoptotic events and for promoting survival of neutrophils 27 . This suggests that CEACAM1-mediated anti-apoptotic events may be important for the resolution of influenza virus infection in vivo, which can be further investigated through infection studies with Ceacam1-knockout mice.
NK cells play a critical role in innate defense against influenza viruses by recognizing and killing infected cells. Influenza viruses, however, employ several strategies to escape NK effector functions, including modification of influenza hemagglutinin (HA) glycosylation to avoid NK activating receptor binding 49 . Homo-or heterophilic CEACAM1 interactions have been shown to inhibit NK-killing 25, 26 , and are thought to contribute to tumor cell immune evasion 50 . Given these findings, one could suggest the possibility that upregulation of CEACAM1 (to inhibit NK activity) may be a novel and uncharacterized immune evasion strategy employed by influenza viruses. Our laboratory is now investigating the role of CEACAM1 in NK cell function. Small-molecule inhibitors of protein kinases or protein phosphatases (e.g. inhibitors for Src, JAK, SHP2) have been developed as therapies for cancer, inflammation, immune and metabolic diseases 51 . Modulation of CEACAM1 phosphorylation, dimerization and the downstream function with small-molecule inhibitors may assist in dissecting the contribution of CEACAM1 to NK cell activity.
The molecular mechanism of CEACAM1 action following infection has also been explored in A549 cells using PR8 virus 52 . Vitenshtein et al. demonstrated that CEACAM1 was upregulated following recognition of viral RNA by RIG-I, and that this upregulation was interferon regulatory factor 3 (IRF3)-dependent. In addition, phosphorylation of CEACAM1 by SHP2 inhibited viral replication by reducing phosphorylation of mammalian target of rapamycin (mTOR) to suppress global cellular protein production. In the present study, we used a more physiologically relevant infection model, primary human ATII cells, to study the role of Further studies will be required to investigate/confirm the molecular mechanisms of CEACAM1 upregulation following influenza virus infection, especially in vivo. As upregulation of CEACAM1 has been observed in other virus infections, such as cytomegalovirus 18 and papillomavirus 19 , it will be important to determine whether a common mechanism of action can be attributed to CEACAM1 in order to determine its functional significance. If this can be established, CEACAM1 could be used as a target for the development of a pan-antiviral agent.
In summary, molecules on the cell surface such as CEACAM1 are particularly attractive candidates for therapeutic development, as drugs do not need to cross the cell membrane in order to be effective. Targeting of host-encoded genes in combination with current antivirals and vaccines may be a way of reducing morbidity and mortality associated with influenza virus infection. Our study clearly demonstrates that increased CEACAM1 expression is observed in primary human ATII cells infected with HPAI H5N1 influenza virus. Importantly, knockdown of CEACAM1 expression resulted in a reduction in influenza virus replication and suggests targeting of this molecule may assist in improving disease outcomes.
Isolation and culture of primary human ATII cells. Human non-tumor lung tissue samples were donated by anonymous patients undergoing lung resection at University Hospital, Geelong, Australia. The research protocols and human ethics were approved by the Human Ethics Committees of Deakin University, Barwon Health and the Commonwealth Scientific and Industrial Research Organisation (CSIRO). Informed consent was obtained from all tissue donors. All research was performed in accordance with the guidelines stated in the National Statement on Ethical Conduct in Human Research (2007) . The sampling of normal lung tissue was confirmed by the Victorian Cancer Biobank, Australia. Lung specimens were preserved in Hartmann's solution (Baxter) for 4-8 hours or O/N at 4 °C to maintain cellular integrity and viability before cells are isolated. Human alveolar epithelial type II (ATII) cells were isolated and cultured using a previously described method 30, 53 with minor modifications. Briefly, lung tissue with visible bronchi was removed and perfused with abundant PBS and submerged in 0.5% Trypsin-EDTA (Gibco) twice for 15 min at 37 °C. The partially digested tissue was sliced into sections and further digested in Hank's Balanced Salt Solution (HBSS) containing elastase (12.9 units/mL; Roche Diagnostics) and DNase I (0.5 mg/mL; Roche Diagnostics) for 60 min at 37 °C. Single cell suspensions were obtained by filtration through a 40 μm cell strainer and cells (including macrophages and fibroblasts) were allowed to attach to tissue-culture treated Petri dishes in a 1:1 mixture of DMEM/F12 medium (Gibco) and small airway growth medium (SAGM) medium (Lonza) containing 5% fetal calf serum (FCS) and 0.5 mg/mL DNase I for 2 hours at 37 °C. Non-adherent cells, including ATII cells, were collected and subjected to centrifugation at 300 g for 20 min on a discontinuous Percoll density gradient (1.089 and 1.040 g/mL). Purified ATII cells from the interface of two density gradients was collected, washed in HBSS, and re-suspended in SAGM medium supplemented with 1% charcoal-filtered FCS (Gibco) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco). ATII cells were plated on polyester Transwell inserts (0.4 μm pore; Corning) coated with type IV human placenta collagen (0.05 mg/mL; Sigma) at 300,000 cells/cm 2 and cultured under liquid-covered conditions in a humidified incubator (5% CO 2 , 37 °C). Growth medium was changed every 48 hours. These culture conditions suppressed fibroblasts expansion within the freshly isolated ATII cells and encouraged ATII cells to form confluent monolayers with a typical large and somewhat square morphology 54 Cell culture and media. A549 carcinomic human alveolar basal epithelial type II-like cells and Madin-Darby canine kidney (MDCK) cells were provided by the tissue culture facility of Australian Animal Health Laboratory (AAHL), CSIRO. A549 and MDCK cells were maintained in Ham's F12K medium (GIBCO) and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO) and maintained at 37 °C, 5% CO 2 .
Virus and viral infection. HPAI A/chicken/Vietnam/0008/2004 H5N1 (H5N1) was obtained from AAHL, CSIRO. Viral stocks of A/Puerto Rico/8/1934 H1N1 (PR8) were obtained from the University of Melbourne. Virus stocks were prepared using standard inoculation of 10-day-old embryonated eggs. A single stock of virus was prepared for use in all assays. All H5N1 experiments were performed within biosafety level 3 laboratories (BSL3) at AAHL, CSIRO.
Cells were infected with influenza A viruses as previously described 6, 29 . Briefly, culture media was removed and cells were washed with warm PBS three times followed by inoculation with virus for 1 hour. Virus was then removed and cells were washed with warm PBS three times, and incubated in the appropriate fresh serum-free culture media containing 0.3% BSA at 37 °C. Uninfected and infected cells were processed identically. For HiSeq analysis, ATII cells from three donors were infected on the apical side with H5N1 at a MOI of 2 for 24 hours in serum-free SAGM medium supplemented with 0.3% bovine serum albumin (BSA) containing 1 mM apocynin dissolved in DMSO or 1% DMSO vehicle control. Uninfected ATII cells incubated in media containing 1% DMSO were used as a negative control. For other subsequent virus infection studies, ATII cells from a different set of three donors (different from those used in HiSeq analysis) or A549 cells from at least three different passages were infected with influenza A viruses at various MOIs as indicated in the text. For H5N1 studies following transfection with siRNA, the infectious dose was optimized to a MOI of 0.01, a dose at which significantly higher CEACAM1 protein expression was induced with minimal cell death at 24 hpi. For PR8 infection studies, a final concentration of 0.5 µg/mL L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) was included in media post-inoculation to assist replication. Virus titers were determined using standard plaque assays in MDCK cells as previously described 55 .
RNA extraction, quality control (QC) and HiSeq analysis. ATII cells from three donors were used for HiSeq analysis. Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen). Influenza-infected cells were washed with PBS three times and cells lysed with RLT buffer supplemented with β-mercaptoethanol (10 μL/mL; Gibco). Cell lysates were homogenized with QIAshredder columns followed by on-column DNA digestion with the RNase-Free DNase Set (Qiagen), and RNA extracted according to manufacturer's instructions. Initial QC was conducted to ensure that the quantity and quality of RNA samples for HiSeq analysis met the following criteria; 1) RNA samples had OD260/280 ratios between 1.8 and 2.0 as measured with NanoDrop TM Spectrophotometer (Thermo Scientific); 2) Sample concentrations were at a minimum of 100 ng/μl; 3) RNA was analyzed by agarose gel electrophoresis. RNA integrity and quality were validated by the presence of sharp clear bands of 28S and 18S ribosomal RNA, with a 28S:18S ratio of 2:1, along with the absence of genomic DNA and degraded RNA. As part of the initial QC and as an indication of consistent H5N1 infection, parallel quantitative real-time reverse transcriptase PCR (qRT-PCR) using the same RNA samples used for HiSeq analysis was performed in duplicate as previously described 6 to measure mRNA expression of IL6, IFNB1, CXCL10, CCL5, TNF, SOCS1 and SOCS3, all of which are known to be upregulated following HPAI H5N1 infection of A549 cells 6 Sequencing analysis and annotation. After confirming checksums and assessing raw data quality of the FASTQ files with FASTQC, RNA-Seq reads were processed according to standard Tuxedo pipeline protocols 56 , using the annotated human genome (GRCh37, downloaded from Illumina iGenomes) as a reference. Briefly, raw reads for each sample were mapped to the human genome using TopHat2, sorted and converted to SAM format using Samtools and then assembled into transcriptomes using Cufflinks. Cuffmerge was used to combine transcript annotations from individual samples into a single reference transcriptome, and Cuffquant was used to obtain per-sample read counts. Cuffdiff was then used to conduct differential expression analysis. All programs were run using recommended parameters. It is important to note that the reference gtf file provided to cuffmerge was first edited using a custom python script to exclude lines containing features other than exon/cds, and contigs other than chromosomes 1-22, X, Y. GO term and KEGG enrichment. Official gene IDs for transcripts that were differentially modulated following HPAI H5N1 infection with or without apocynin treatment were compiled into six target lists from pairwise comparisons ("ND vs. HD Up", "ND vs. HD Down", "ND vs. HA Up", "ND vs. HA Down", "HD vs. HA Up", "HD vs. HA Down"). Statistically significant differentially expressed transcripts were defined as having ≥2-fold change with a Benjamini-Hochberg adjusted P value < 0.01. A background list of genes was compiled by retrieving all gene IDs identified from the present HiSeq analysis with FPKM > 1. Biological process GO enrichment was performed using Gorilla, comparing unranked background and target lists 57 . Redundant GO terms were removed using REVIGO 58 . Target lists were also subjected to KEGG pathway analysis using a basic KEGG pathway mapper 59 and DAVID Bioinformatics Resources Functional Annotation Tool 60,61 .
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). mRNA concentrations of genes of interest were assessed and analyzed using qRT-PCR performed in duplicate as previously described 6 . Briefly, after total RNA extraction from influenza-infected cells, cDNA was SCIEntIfIC RepoRtS | (2018) 8:15468 | DOI:10.1038/s41598-018-33605-6 prepared using SuperScript ™ III First-Strand Synthesis SuperMix (Invitrogen). Gene expression of various cytokines was assessed using TaqMan Gene Expression Assays (Applied Biosystems) with commercial TaqMan primers and probes, with the exception of the influenza Matrix (M) gene (forward primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′; probe 5′-FAM-TCAGGCCCCCTCAAAGCCGAG-NFQ-3′) 62 . Specific primers 63 (Table S5) were designed to estimate the expression of CEACAM1-4L, -4S, -3L and -3S in ATII and A549 cells using iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer's instruction. The absence of nonspecific amplification was confirmed by agarose gel electrophoresis of qRT-PCR products (15 μL) (data not shown). Gene expression was normalized to β-actin mRNA using the 2 −ΔΔCT method where expression levels were determined relative to uninfected cell controls. All assays were performed in duplicate using an Applied Biosystems ® StepOnePlus TM Real-Time PCR System. Western blot analysis. Protein expression of CEACAM1 was determined using Western blot analysis as previously described 6 . Protein concentrations in cell lysates were determined using EZQ ® Protein Quantitation Kit (Molecular Probes TM , Invitrogen). Equal amounts of protein were loaded on NuPAGE 4-12% Bis-Tris gels (Invitrogen), resolved by SDS/PAGE and transferred to PVDF membranes (Bio-Rad). Membranes were probed with rabbit anti-human CEACAM1 monoclonal antibody EPR4049 (ab108397, Abcam) followed by goat anti-rabbit HRP-conjugated secondary antibody (Invitrogen). Proteins were visualized by incubating membranes with Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific) followed by detection on a Bio-Rad ChemiDoc ™ MP Imaging System or on Amersham ™ Hyperfilm ™ ECL (GE Healthcare).
To use β-actin as a loading control, the same membrane was stripped in stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) and re-probed with a HRP-conjugated rabbit anti-β-actin monoclonal antibody (Cell Signaling). In some cases, two SDS/PAGE were performed simultaneously with equal amounts of protein loaded onto each gel for analysis of CEACAM1 and β-actin protein expression in each sample, respectively. Protein band density was quantified using Fiji software (version 1.49J10) 64 . CEACAM1 protein band density was normalized against that of β-actin and expressed as fold changes compared to controls.
Knockdown of endogenous CEACAM1. ATII and A549 cells were grown to 80% confluency in 6-well plates then transfected with small interfering RNA (siRNA) targeting the human CEACAM1 gene (siCEACAM1; s1976, Silencer ® Select Pre-designed siRNA, Ambion ® ) or siRNA control (siNeg; Silencer ® Select Negative Control No. 1 siRNA, Ambion ® ) using Lipofetamine 3000 (ThermoFisher Scientific) according to manufacturer's instructions. Transfection and silencing efficiency were evaluated after 48 hours by Western blot analysis of CEACAM1 protein expression and by qRT-PCR analysis of CEACAM1 variants. In parallel experiments, virus replication and cytokine/chemokine production was analyzed in siCEACAM1-or siNeg-transfected cells infected with H5N1 virus (MOI = 0.01) at 24 hpi. Statistical analysis. Differences between two experimental groups were evaluated using a Student's unpaired, two-tailed t test. Fold-change differences of mRNA expression (qRT-PCR) between three experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple-comparison test. Differences were considered significant with a p value of <0.05. The data are shown as means ± standard error of the mean (SEM) from three or four individual experiments. Statistical analyses were performed using GraphPad Prism for Windows (v5.02).
All data generated or analyzed during this study are included in this published article or the supplementary information file. The raw and processed HiSeq data has been deposited to GEO (GSE119767; https://www.ncbi. nlm.nih.gov/geo/). | How do influenza viruses escape binding by the natural killer cell activating receptors? | {
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2,250 | Improved Pharmacological and Structural Properties of HIV Fusion Inhibitor AP3 over Enfuvirtide: Highlighting Advantages of Artificial Peptide Strategy
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541410/
SHA: f2fcc16391f946c99717b63ec9a24e5384aac381
Authors: Zhu, Xiaojie; Zhu, Yun; Ye, Sheng; Wang, Qian; Xu, Wei; Su, Shan; Sun, Zhiwu; Yu, Fei; Liu, Qi; Wang, Chao; Zhang, Tianhong; Zhang, Zhenqing; Zhang, Xiaoyan; Xu, Jianqing; Du, Lanying; Liu, Keliang; Lu, Lu; Zhang, Rongguang; Jiang, Shibo
Date: 2015-08-19
DOI: 10.1038/srep13028
License: cc-by
Abstract: Enfuvirtide (T20), is the first HIV fusion inhibitor approved for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, its clinical application is limited because of short half-life, drug resistance and cross-reactivity with the preexisting antibodies in HIV-infected patients. Using an artificial peptide strategy, we designed a peptide with non-native protein sequence, AP3, which exhibited potent antiviral activity against a broad spectrum of HIV-1 strains, including those resistant to T20, and had remarkably longer in vivo half-life than T20. While the preexisting antibodies in HIV-infected patients significantly suppressed T20’s antiviral activity, these antibodies neither recognized AP3, nor attenuated its anti-HIV-1 activity. Structurally different from T20, AP3 could fold into single-helix and interact with gp41 NHR. The two residues, Met and Thr, at the N-terminus of AP3 form a hook-like structure to stabilize interaction between AP3 and NHR helices. Therefore, AP3 has potential for further development as a new HIV fusion inhibitor with improved antiviral efficacy, resistance profile and pharmacological properties over enfuvirtide. Meanwhile, this study highlighted the advantages of artificially designed peptides, and confirmed that this strategy could be used in developing artificial peptide-based viral fusion inhibitors against HIV and other enveloped viruses.
Text: The sequences of gp41 NHR-or CHR-derived peptides. The residues corresponding to the NHR pocket region are marked in red. The residues for the PBD are marked in blue, and the MT-hook residues adjacent to the N terminus of PBD are marked in green. 5HRu peptide consists of 5 copies of artificial sequence template (AEELAKK) underlined. The mutant residues in PBD of AP2 and AP3 were highlighted in pink. (b) The inhibitory activity of AP1, AP2, AP3 and T20 on infection by HIV-1 IIIB (subtype B, X4) in MT-2 cells (left panel) by HIV-1 Bal (subtype B, R5) in M7 cells (right panel). Each sample was tested in triplicate and the experiment was repeated twice. The data are presented as means ± SD.
Scientific RepoRts | 5:13028 | DOi: 10 .1038/srep13028
To address these obstacles, many efforts have been made to optimize T20 and gp41 CHR-derived peptides. Some of these peptides have better inhibitory activities against T20-resistant strains and/or longer half-life than T20. However, they still have the problem to cross-react with the preexisting antibodies in the sera of HIV-infected patients because they contain some native CHR sequences. Based on the universal artificial peptide template of 5HRu, we previously designed the artificial peptides of AP1 (PBD-m4HR) and AP2 (PBDtrp-m4HR), and have made preliminary research on their inhibitory activity against HIV-1 Env-mediated cell-cell fusion 16 . In the present study, we designed a new artificial peptide, AP3 (Fig. 1a) , aiming to apply the "M-T hook" structure to stabilize the interaction of the artificial peptide with the hydrophobic pocket on the gp41 NHR trimer 17, 18 . After comprehensively studying its antiviral activity, biochemical property, crystal structure, functional mechanism, in vivo half-life and, for the first time, the effect of preexisting antibodies in the sera of HIV-infected patients, we found that the newly designed artificial peptide, AP3, exhibited improved antiviral activity, drug resistance profile and pharmacological properties over T20. Particularly, the preexisting antibodies in the sera of HIV-infected patients did not suppress, but enhanced the anti-HIV-1 activity of AP3. These results suggest that AP3 has potential for development as a new anti-HIV drug and confirm that this strategy can be used for designing artificial antiviral peptides against other enveloped viruses, such as SARS-CoV 19 , MERS-CoV 20 , and paramyxovirus 21 .
AP3 inhibited HIV-1 infection with higher potency than T20. Our previously designed artificial peptides AP1 and AP2 could inhibit HIV-1 Env-mediated cell-cell membrane fusion 16 . He and colleagues reported that adding two amino acids of Met and Thr to the N-terminus of a CHR-peptide could enhance their anti-HIV-1 activity 17, 18 . Here we designed a new artificial peptide, AP3, by adding Met and Thr to the N-terminus of AP2 (Fig. 1a) . We then compared AP3 with AP1, AP2 and T20 for their anti-HIV-1 activity against divergent HIV-1 strains, including the laboratory-adapted viruses, IIIB (subtype B, X4) and Bal (subtype B, R5), and a series of primary HIV-1 isolates, as well as the T20-resistant strains. As shown in Fig. 1b , AP3 exhibited higher inhibitory activities on infection by HIV-1 IIIB and HIV-1 Bal strains (IC 50 : 3.06 and 15.09 nM, respectively) than AP1 (IC 50 : 86.25 and 396.14 nM, respectively), AP2 (IC 50 : 23.05 and 49.95 nM, respectively), and T20 (IC 50 : 13.63 and 30.21 nM, respectively). The inhibitory activity of AP3 on infection by divergent primary HIV-1 isolates with distinct genotypes (subtypes A -E and group O) and phenotypes (R5 and X4) was also higher than that of AP2 and T20 (Table 1) . While T20 was not effective against T20-resistant HIV-1 strains at the concentration as high as 2,000 nM, AP3 could effectively inhibit infection of these strains with IC 50 in the range of 13 ~ 90 nM, which was about 2-to 4-fold more effective than AP2 (Table 1 ). These results indicate that the artificial peptide AP3 has remarkably improved anti-HIV-1 activity against a broad spectrum of HIV-1 strains, including T20resistant variants, over T20 and the artificial peptides AP1 and AP2. The preexisting antibodies in HIV-1-infected patients neither recognized AP3, nor attenuated its anti-HIV-1 activity. Previous studies have shown that the preexisting antibodies in HIV-1-infected patients, including those cross-reacting with T20 and those specific for the binding sites of T20 in gp120 (e.g., the C1 and V3 loop regions) and gp41 (e.g., the NHR domain), could significantly block the fusion inhibitory activity of T20 14, 15 . Here we investigated the influence of preexisting antibodies against AP3 peptide. As shown in Fig. 2a , both T20 and C46 reacted with the antibodies in sera from five HIV-1-infected patients; however, none of the three artificial peptides AP1, AP2 and AP3 was recognized by the preexisting antibodies. The inhibitory activity of T20 on HIV-1 IIIB infection was reduced about 1.9-fold to > 3.6-fold in the presence of the sera from HIV-1-infected patients ( Fig. 2b and Supplementary Table S1), confirming that the preexisting antibodies in sera of HIV/AIDS patients can attenuate the anti-HIV-1 activity of T20 14, 15 . However, none of the artificial peptides in the present study showed significant decrease of anti-HIV-1 activity in the presence of patients' sera. Instead, the antiviral activity of AP3 increased in the presence of antisera from HIV-1-infected patients ( Fig. 2b and Supplementary Table S1), suggesting that anti-HIV-1 antibodies actually enhanced the anti-HIV-1 activity of AP3, possibly because the binding of the antibodies to some sites in gp120 or gp41 promote the interaction of AP3 with viral gp41 NHR region.
AP3 had longer half-life than T20. Although T20 has shown efficacy in inhibiting HIV-1 infection, its major weakness lies in its short half-life in plasma (about 2 h) [22] [23] [24] . As a result, T20 has to be administered subcutaneously twice daily at 90 mg per dose, often causing serious injection-site reactions 25, 26 . Here, we performed pharmacokinetic studies by intravenous administration of AP3, AP2, and T20, respectively, to SD rat at a dose of 1 mg/kg, in order to compare their in vivo circulation time. As expected, T20 exhibited a shorter half-life and lower AUC (0-t) from systemic circulation, while AP3 and AP2 demonstrated much higher concentration and longer circulation time ( Table 2 ). The pharmacokinetic profiles of AP3 and AP2 fit a non-compartment model. The pharmacokinetic parameters were calculated with PK Solver. The in vivo elimination half-life of AP3 (t 1/2 = 6.02 h) was about 2.8-fold longer than that of T20 (t 1/2 = 1.57 h). This result provided the theoretical basis for reducing the injection frequency and dose of the fusion inhibitor, in conjugation with the improved antiviral potency of AP3. Therefore, replacement of T20 with AP3 may significantly reduce injection-site reactions and the drug cost, which would promote the clinical applications of the HIV fusion inhibitor in resource-poor regions or countries.
AP3 was much more resistant than T20 to proteolytic degradation by proteinase K and rat liver homogenate. We compared the stability of T20 and AP3 in the presence of proteinase K (a broad-spectrum serine proteinase) and rat liver homogenate. After treatment with 20 ng/mL of proteinase K for 2 h at 37 °C, only 29% of the parental T20 peptide remained, as detected by LC-MS analysis. Under the same condition, AP3 retained 100% of its prototype (Fig. 3a ). In addition, AP3 showed a significantly enhanced in vitro metabolic stability over T20 in the presence of liver homogenate (Fig. 3b) .
These results indicate that the artificial peptide AP3 is much more resistant to proteolytic degradation than the natural peptide T20, which may contribute to its significant longer in vivo half-life than T20 as described above.
AP3 formed stable α-helical complex and block gp41 6-HB formation. To investigate the antiviral mechanism of AP3, the thermal stability of AP3/N36 complex was compared with that of AP1/N36, AP2/N36, T20/N36, and C34/N36 complexes by circular-dichroism (CD) spectroscopy 27 . Because T20 lacks the pocket-binding domain (PBD), the T20/N36 complex did not show a typical α -helical conformation, in consistence with our previous studies 8, 9 . Similar to the α -helicity of C34/N36 complex 3 , the AP1/N36, AP2/N36 and AP3/N36 complexes all formed a saddle-shaped negative peak at 208 nm and 222 nm, indicating their α -helical structures (Fig. 4a) Fig. 4b) , indicating that the α -helical complex formed by AP3 and N36 is the most stable among the four complexes.
Then we compared the inhibitory activity of AP3 with that of AP1 and AP2 on 6-HB formation between C34 and N36. Since T20 cannot block 6-HB formation 8, 9 , we used a small-molecule HIV-1 fusion inhibitor, ADS-J1 28, 29 , to replace T20 as a control of 6-HB inhibition. As expected, ADS-J1 could effectively inhibit 6-HB formation with IC 50 of 2.75 μ M 8, 9, [27] [28] [29] . AP3 was highly effective against 6-HB formation in a dose-dependent manner with an IC 50 value of 0.24 μ M, about 30-and 15-fold more potent than AP1 and AP2, respectively (Fig. 4c) , confirming that AP3 can potently block gp41 6-HB fusion core formation, thus inhibiting HIV-1 fusion with the target cell membrane.
Structural basis for the potent fusion inhibitory activity of the artificial peptide AP3. To elucidate the molecular determinants of these artificial peptides, we successfully solved all three complex structures of AP1/AP2/AP3 peptides binding with gp41 NHR. For AP1 and AP2, an optimized linker Each sample was tested in triplicate and the experiment was repeated twice. The data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. "SGGRGG" was used to assemble the NHR and the artificial peptide into a single recombinant protein (N36-L6-AP1 or N36-L6-AP2). However, a similar strategy failed on the crystallization of AP3; therefore, we decided to cocrystallize the synthetic peptide N45 and AP3 peptide, and eventually the complex crystals were obtained. Interestingly, the crystals of three different inhibitors belong to three distinctive space groups: P2 1 for N36-L6-AP1, R32 for N36-L6-AP2, and P6 3 for N45/AP3. As expected, the NHR portions in three structures all form a trimeric core, while the AP1, AP2 or AP3 portion folds into a single-helix conformation and binds to NHR-trimer to form a typical 6-HB, similar to that of the HIV-1 gp41 core structure formed by the native CHR peptide C34 and N36 (Fig. 5a) . Also, the conserved hydrophobic residues, such as W43, W46 and I50, in the artificial peptides were deeply buried into the hydrophobic Table 2 . Pharmacokinetic parameters of AP2, AP3 and T20 following intravenous administration at 1 mg/kg in male SD rats (n = 2). Figure 3 . Sensitivity of AP3 and T20 to proteolytic degradation by proteinase K and rat liver homogenate. (a) After digestion by proteinase K at pH 7.2 and (b) rat liver homogenate, the residual amount of AP3 and T20 was detected by LC-MS analysis. The experiment was performed in triplicate and the data are presented as means ± SD. The inhibition of AP1, AP2, AP3, T20 and ADS-J1 against 6-HB formation between N36 and C34 was detected by ELISA using the 6-HB-specific mAb NC-1. Each sample was tested in triplicate, and the data are presented as means ± SD. grooves formed between each pair of NHR helices, similar to the corresponding residues of W628, W631 and I635 in the native gp41 CHR (Fig. 5b) . AP peptides exhibited better affinity against gp41 natural CHR. In C34, which contains the natural CHR sequence from W628 to L661, no strong interaction between I642 and Q565 in the viral gp41 NHR-CHR complex was found (Fig. 5c) . However, in the corresponding sequence (from W43 to K76) of AP1 and AP2, a hydrogen bond was established between S57 (corresponding to I642 in CHR) and Q18 (corresponding to Q565 in NHR) in N36-L6-AP1, N36-L6-AP2 and N45/AP3. Thus, S57 in AP1/AP2/AP3 plays a role in stabilizing the interactions between the artificial peptide inhibitor and its NHR target, resulting in their stronger binding affinity. Moreover, in NHR-CHR, L567 and L568 on two adjacent NHRs form a hydrophobic groove, in which T639 is buried (Supplementary Fig. S1a ). However, in N36-L6-AP1, N36-L6-AP2 and N45/AP3, I54 (corresponding to T639 in CHR) can strongly bind to L20 and L21 through fully hydrophobic side chain interactions. Similarly, the interaction of I64 (corresponding to S659 in CHR) with L10 and L11 (corresponding to L567 and L568 in NHR, respectively) in N36-L6-AP1, N36-L6-AP2 and N45/AP3 has been significantly enhanced ( Supplementary Fig. S1b ).
Like the gp41 CHR helix, the helices of AP1, AP2 and AP3 also have two different sides, a hydrophobic side facing toward the NHR and a hydrophilic one facing outward. It is expected that the enhancement of the hydrophilicity of the exposed side of the inhibitors can increase their antiviral activity and solubility. To achieve this goal, the amino acid residues with hydrophobicity, or low hydrophilicity, like N637, S640, L641 and S644 in CHR, were changed to the amino acid residues with high hydrophilicity, like E52, K55, K56 and E59 in AP1, AP2 and AP3, respectively. Moreover, the hydrophobic residue M629 in CHR was replaced with a hydrophilic residue E44 in AP2 and AP3 ( Supplementary Fig. S2 ). These hydrophilic residues, such as glutamic acid and lysine, can increase the solubility of whole peptide and, hence, stabilize the complex formed by the inhibitor and its target. It has been proved that the EE-KK double salt bridge can stabilize helix conformation 30 . We have identified this kind of interaction between i and i + 3 or i + 4 positions on the three complex structures. In N36-L6-AP1, R48 interacts with E45 and E52 to form a salt bridge network. In N36-L6-AP2, E45 interacts with K48, and E52 binds to K56, while in N45/AP3, K69 binds to E66 ( Supplementary Fig. S2 ). These strong salt bridges formed by the oppositely charged residues stabilize AP peptide conformation, bringing its inhibitory effect into full play.
As previously reported, addition of the "M-T hook" to the CHR peptides C34 and sifuvertide could dramatically improve the anti-HIV-1 activity 17, 18 . As expected, the N-terminal Met and Thr of AP3 forms a hook-like structure (Fig. 5d) . The hydrophobic methionine side chain of M41 accommodates the groove between AP3 and NHR helices, capping the hydrophobic pocket. This interaction leads to a series of conformational changes. The main chain of AP3 at W43 moves 1.91 Å closer to NHR compared to AP2 (Supplementary Fig. S3 ). The side chain of W43 in AP3 flips around 90 degrees and is buried deeper than that of AP2. The side chain of E44 turns back to interact to D47, but the E45 side chain turns back from K48 and interacts with T42. Therefore, this M-T hook structure could further stabilize the binding between AP3 and NHR target.
Enfuvirtide, also known as T20, was approved by the U.S. FDA as the first HIV entry inhibitor-based antiviral drug for use with other anti-HIV medicines to treat HIV-1 infected adults and children at ages 6-16 years 23,31,32 (http://www.fuzeon.com). Although T20 is an indispensable anti-HIV drug for HIV/ AIDS patients who have failed to respond to the current antiretroviral therapeutics, its shortcomings have limited its clinical application. T20 has lower anti-HIV activity and shorter half-life than other CHR peptides containing PBD, such as C34 and C38 8, 9, 33 . In addition, T20-resistant HIV-1 variants emerged shortly (e.g., 14 days) after its use in patients 34 . Most of the T20-resistant viruses carried mutations in the GIV motif (residues 36-45: GIVQQQNNLL) in the gp41 NHR domain 10, [34] [35] [36] [37] [38] . The lack of PBD contributes to the major weaknesses of T20 described above. Since the conserved hydrophobic pocket in the gp41 NHR-trimer plays a critical role in stabilizing the interaction between the gp41 NHR and CHR and formation of the fusogenic 6-HB core 1, 39, 40 , the PBD-containing CHR-peptide, like C34, can bind to viral gp41 trimer more strongly and stably, thus possessing more potent anti-HIV activity than T20, a CHR peptide without PBD 8, 9 . In the absence of PBD, T20 mainly interacts with the middle region of the NHR domain containing the GIV motif. Therefore, a virus with mutations in this motif is generally resistant to T20 10, [34] [35] [36] [37] [38] . Compared with other anti-HIV drugs, another weakness of T20 is its cross-reactivity with the preexisting antibodies in HIV-1-infected patients. Besides gp41, T20 could also bind to some regions in gp120. The preexisting antibodies specific for the T20's binding sites in gp120 and gp41 may indirectly suppress the anti-HIV activity of T20 14, 15 .
Addition of PBD to the N-terminus of T20, such as T-1249, could significantly improve the anti-HIV-1 potency, half-life and drug-resistance profile 33, [41] [42] [43] . Addition of M-T hook structure to the N-terminus of a PBD-containing CHR-peptides, such as MT-C34 or MT-SFT, could further increase the anti-HIV-1 activity of the corresponding CHR-peptides 17, 18 . Deletion of the GIV-motif-binding domain from a CHR-peptide, such as CP621-652 and CP32M, is another effective approach to increase the genetic barrier to drug resistance 44, 45 . However, none of the above approaches is effective in preventing the cross-reaction of T20 with the preexisting anti-gp41 antibodies in HIV/AIDS patients, since the above-modified peptides mainly contain the native sequences of the HIV-1 gp41 CHR domain. Our previous studies have shown that AP1 and AP2, artificial peptides with non-native protein sequences, could form coiled-coil structure to interact with gp41 NHR and inhibit HIV-1 Env-mediated cell-cell fusion 16 . In the present study, we designed a new artificial peptide, AP3, by adding M-T hook structure to the N-terminus of AP2 (Fig. 1a) , followed by investigating the influence of preexisting anti-gp41 antibodies in HIV-infected patients on AP3, using AP1, AP2 and T20 as controls. We demonstrated that sera of HIV-infected patients could bind to T20 and significantly reduce its potency against HIV-1. However, these same serum samples did not interact with the three artificial peptides and hardly impaired their antiviral activity. Surprisingly, the antibodies in the sera could even enhance AP3's anti-HIV-1 activity (Fig. 2a,b and Supplementary Table S1 ). These results confirmed, for the first time, that replacement of the native viral sequence in T20 with an artificial sequence is an effective approach to overcome a key shortcoming of T20 whereby its anti-HIV activity could be attenuated by preexisting anti-gp41 antibodies in HIV/AIDS patients. It is worthwhile to explore why the antibodies in the sera is able to enhance the anti-HIV-1 activity of AP3. Our recent study has demonstrated that T20's anti-HIV-1 activity is enhanced by a non-neutralizing antibody directed against the NHR domain of the HIV-1 gp41 46 . We thus hypothesize that some of the anti-gp41 antibodies in HIV/AIDS patients may bind to a site in NHR domain adjacent to the AP3's binding region, resulting in increased interaction between AP3 and NHR-trimer and enhanced antiviral activity of AP3.
We then compared the inhibitory activity of AP3 with M-T hook and T20/AP2 without M-T hook on infection by divergent HIV-1 strains. AP3 was more effective than either AP2 or T20 in inhibiting infection by the laboratory-adapted strains and the primary isolates of HIV-1, including those resistant to T20 (Fig. 1b, Table 1 ). One may question whether AP3 can also induce drug-resistant viruses in patients if it is used in clinics to treat HIV-infected patients. We believe that AP3 is expected to have much higher genetic barrier to resistance than T20 because AP3 contains PBD, while T20 lacks PBD. Dwyer et al. 33 used T2544, a PBD-containing CHR-peptide, to carry out a passaging experiment, using T20 as a control. They demonstrated that T20 could induce a mutant virus with high resistance (81-fold) to T20 in about 1 month, while T2544 failed to induce a resistant strain in more than 2 months in culture. After extending the passaging experiment for almost 8 months, they identified one strain with a weak resistance (8.3-fold) to T-2544, and the related mutation sites were not in the gp41 pocket region, suggesting that the PBD-containing CHR-peptides, including AP3, may have difficulty to induce drug-resistance.
AP3 also had longer half-life than T20 (Table 2) , possibly because the artificial peptide AP3 is less sensitive to the proteolytic enzymes than T20 with native viral protein sequence. Removal of the proteolytic enzymes' cleavage sites in AP3 peptide is expected to further extend its half-life. These results confirmed that replacement of native protein sequence with artificial sequence and addition of the M-T hook to the PBD-containing peptide is a sound strategy for designing HIV fusion inhibitory peptides with improved antiviral activity and pharmacological properties when compared to T20.
Since the three-dimensional structures of AP peptides had not been investigated before the present study, the optimization of these artificial peptide inhibitors could not be performed rationally. Our structural studies of the artificial peptides AP1/AP2/AP3 in complex with NHR showed that AP peptides, just like the CHR peptide C34, could bind to gp41 NHR to form a canonical 6-HB structure (Fig. 5a) . It is well known that a deep hydrophobic pocket exists in each groove on the surface of the viral gp41 NHR trimer. The hydrophobic residues I635, W631 and W628 in the gp41 CHR bind with the hydrophobic residues in the wall of this pocket, resulting in the formation of stable 6-HB by the strong interaction between CHR and NHR. This important feature has been well preserved in the AP1/AP2/AP3 6-HB structures (Fig. 5b) , which may account for the potent HIV-1 fusion inhibitory activities of these artificial peptides.
A new hydrogen bond, which was established between S57 and Q18 in AP1/AP2/AP3 complexes, does not exist in the viral gp41 CHR-NHR complex, suggesting that S57 may play an important role in stabilizing the interactions between the peptide and NHR, resulting in binding affinities of AP1/AP2/AP3 that are stronger than those of HIV-1 gp41 CHR to NHR. Furthermore, the EE-KK double salt bridge formed between the i and i + 4 positions in the AP1/AP2/AP3 structures could stabilize helix conformation and increase the inhibitory effect of these peptides. Compared with AP1, triple-site mutations were introduced in AP2 and AP3, i.e. M44E, R48K and E49K. Those substitutions not only increase solubility of the peptide, but also trigger a series of rearrangements of certain intrahelical salt bridges to improve the stability of CHR helix structure and HIV-1 fusion inhibitory activity.
M-T hook was previously demonstrated to be an effective step toward increasing the stable interaction between a CHR-peptide and the HIV-1 gp41 pocket 17, 18 . Therefore, AP2 was further optimized by incorporating Met and Thr at its N-terminus. CD spectroscopy and thermal denaturation results both indicate that the incorporation of M-T hook contribute to the formation of a more stable 6-HB core structure between AP3 (M-T hook-optimized AP2) and N36. In addition, the EE-KK double salt bridge formed between i and i + 4 positions in the N36-L6-AP3 structure contributed to increased CHR helix and 6-HB stability, resulting in improved potency of AP3, as has been noted in studies of CHR-peptides with EE-KK double mutations 30, 33, 47, 48 . Also, the HIV-1 fusion activity and half-life of AP2 may have been strengthened and extended, respectively, by the addition of M-T hook in the design of AP3.
In conclusion, AP3, an artificial peptide with both PBD and M-T hook structures, exhibited improved anti-HIV-1 activity and drug-resistance profile, as well as prolonged half-life. Moreover, it did not react with the preexisting antibodies in the sera of HIV/AIDS patients. Consequently, its antiviral activity Scientific RepoRts | 5:13028 | DOi: 10.1038/srep13028 was not significantly affected by these antibodies. Therefore, AP3 shows promise as a candidate for further development as a new HIV fusion inhibitor for clinical use. This study also provides important structure and activity information for the rational design of novel artificially peptide inhibitors. Besides, our results highlighted the advantages of artificially designed peptides and confirmed that this strategy could be widely used in development of artificial peptide-based virus fusion inhibitors against HIV-1 and other enveloped viruses with class I membrane fusion proteins, such as SARS-CoV 19 , MERS-CoV 20 , and paramyxovirus 49 .
Ethics statement. This study did not involve human experimentation; the only human materials used were serum samples obtained from HIV-1-infected individuals with the approval by the Ethics Committee of the Shanghai Public Health Clinical Center, Fudan University (Protocol No. SPHCC-125-2). The methods were carried out in accordance with the approved guidelines. All of these sera samples came from adults; no minor was involved in this study. Written informed consent for the use of the clinical specimens was obtained from all patients involved in this study.
Peptide synthesis. A panel of peptides (Fig. 1a) , including T20, C34, C46, AP1, AP2, AP3, as well as NHR-derived N-peptides, N36 and N45, were synthesized with a standard solid-phase FMOC method, as described previously 8, 50 . All peptides were acetylated at the N terminus and amidated at the C terminus. The peptides were found to be about 95% pure by HPLC and were identified by mass spectrometry (Perseptive Biosystems, Framingham, MA, USA). Concentrations of the peptides were determined by UV absorbance and a theoretically calculated molar-extinction coefficient based on tryptophan and tyrosine residues.
Qualification assay. Chromatographic analyses were performed using an ODS-C8 column (5 μ m, 100 mm × 2.0 mm ID) kept at ambient temperature. The mobile phase was composed of acetonitrile-water-formic acid in the ratio of 50:50:0.1 (v/v/v) at a flow rate of 0.3 mL/min. The sample injection volume was 10 μ L. Acetonitrile was HPLC grade, and other chemical reagents and solvents were analytical grade. A Thermo TSQ Quantum Discovery MAX triple-quadruple tandem mass spectrometer equipped with ESI source (San Jose, CA) and Surveyor LC pump were used for LC-MS analysis. Data acquisition and data processing were performed by using Xcalibur software and LCQuan 2.0 data analysis program (Thermo Finnigan), respectively. Optimized MS parameters were as below: 4800 V spray voltage, 40.0 psi sheath gas pressure, 1.0 psi auxiliary valve flow, and 300 °C of capillary temperature. When running collision-induced dissociation (CID), the pressure was set to 1.5 mTorr. The selected reaction monitoring (SRM) mode was used for AP3 while the selected ion monitoring (SIM) mode was preformed for T20. The following transitions were recorded: m/z 670.5 for AP3, m/z 1498.6 for T20. The masses of synthetic peptides T20, AP1, AP2 and AP3 were determined by MALDI-TOF-MS (Supplementary Fig. S4 and S5 ).
Expression and purification of fusion protein N36-L6-AP1 and N36-L6-AP2. Using overlapping PCR, the DNA fragment encoding AP1 or AP2 peptide was attached to the 3′-end of the cDNA of gp41 NHR ("N36", 546-581), with a short linker ("L6", SGGRGG) between them. Then, the whole sequence was subcloned into the pET-28a vector (Novagen, USA) with an artificial SUMO-tag between the N-terminal His-tag and the target protein. The pET-28a-SUMO-N36-L6-AP1-or pET-28a-SUMO-N36-L6-AP2-transformed E. coli cells were induced by adding 1 mM IPTG and incubating overnight at 16 °C. Fusion protein was purified by Ni-NTA affinity resin (Qiagen, Valencia, CA, USA), and the His-SUMO-tag was cleaved off by Ulp1 enzyme treatment at 4 °C for 2 h. The purified N36-L6-AP1 or N36-L6-AP2 was applied onto a Superdex-75 gel filtration column (GE Healthcare, Piscataway, NJ, USA). Fractions containing N36-L6-AP1 or N36-L6-AP2 trimer were collected and concentrated to different concentrations by ultrafiltration.
Crystallization, data collection, and structure determination. The fusion protein N36-L6-AP1 was crystallized at 16 °C using the hanging drop, vapor-diffusion method. The drops were set on a siliconized cover clip by equilibrating a mixture containing 1 μ l protein solution (25 mg/ml N36-L6-AP1 trimer in 20 mM Tris-HCl pH 8.0 and 150 mM NaCl) and 1 μ l reservoir solution (0.1 M Tris-HCl pH 8.5, 32% (w/v) PEG3350, and 0.2 M MgCl 2 ) against a 400 μ l reservoir solution. After one week, single crystals formed and were flash frozen by liquid nitrogen for future data collection. Fusion protein N36-L6-AP2 was crystallized in a similar way with a different reservoir solution (0.1 M Tris-HCl pH 8.0, 34% (w/v) PEG3350, and 0.2 M MgCl 2 ). To obtain the complex crystal of AP3 and NHR, synthesized AP3 was first mixed with peptide N45 at 1:1 molar ratio and then applied onto a Superdex-75 gel filtration column (GE Healthcare, Piscataway, NJ, USA) to isolate the formed 6-HB. Fractions containing N45/AP3 trimer were collected and concentrated to 30 mg/ml, then crystallized at 16 °C using the hanging drop, vapor-diffusion method.The drops were set on a siliconized cover clip by equilibrating a mixture containing 1 μ l protein solution (20 mM Tris-HCl pH 8.0 and 150 mM NaCl) and 1 μ l reservoir solution (0.2 M Ammonium Sulfate, 0.1 M Bis-Tris pH 6.5, and 25% w/v PEG 3350) against a 400 μ l reservoir solution. After 3 days, single crystals formed and were flash frozen by liquid nitrogen for future data collection. The datasets of N36-L6-AP1 were collected at 100 K at beamline 19-ID of the Advanced Photon Source (Argonne National Laboratory, USA). The datasets of N36-L6-AP2 were collected on an in-house x-ray source (MicroMax 007 x-ray generator, Rigaku, Japan) at the Institute of Biophysics, ChineseAcademy of Sciences. The datasets of AP3/N45 complex crystals were collected at beamline BL-19U1 of the Shanghai Synchrotron Radiation Facility, China. X-ray diffraction data were integrated and scaled using the HKL2000 program 51 . The phasing problem of all three structures was solved by the molecular replacement method using PHENIX.phaser 52 with a crystal structure of HIV gp41 NHR-CHR (PDB entry: 1SZT) as a search model. The final models were manually adjusted in COOT 53 and refined with PHENIX.refine 54 . All coordinates were deposited in the Protein Data Bank (N36-L6-AP1: 5CMU; N36-L6-AP2: 5CN0; and N45/AP3: 5CMZ). The statistics of data collection and structure refinement are given in Supplementary Table S2 .
Determination of the cross-reactivity of the native and artificial peptides with the preexisting antibodies in HIV-1-infected patients by sandwich ELISA. A sandwich ELISA was conducted to determine the cross-reactivity of the peptides with the preexisting antibodies in HIV-1-infected patients. T20, C46, AP1, AP2 and AP3 were coated onto the wells of 96-well polystyrene plates (Costar, Corning Inc., Corning, NY) at 10 μ g/ml. The wells were then blocked with 1% gelatin, followed by addition of 50 μ l of serially diluted sera from HIV-1-infected patients and incubation at 37 °C for 1 h. Then, HRP-labeled goat-anti-human IgG (Abcam, UK) and TMB were added sequentially. A450 was determined with an ELISA reader (Ultra 384, Tecan).
patients. Inhibition of peptides on HIV-1 IIIB (subtype B, X4)infection in the presence of HIV-1-infected patients' sera was determined as previously described 55 . Briefly, each peptide was mixed with serially diluted serum from an HIV-1-infected patient at room temperature for 30 min. Next, the mixture of peptide/serum and HIV-1 (100 TCID 50 ) were added to MT-2 cells (1 × 10 5 /ml) in RPMI 1640 medium containing 10% FBS. After incubation at 37 °C overnight, the culture supernatants were replaced with fresh culture medium. On the fourth day post-infection, culture supernatants were collected for detection of p24 antigen by ELISA.
CD Spectroscopy and Thermal Midpoint Analysis. The secondary structure of AP1, AP2 or AP3 peptides mixed with N36 was analyzed by CD spectroscopy as previously described 56 . Briefly, each peptide or peptide mixture was dissolved in phosphate-buffered saline (PBS: 50 mM sodium phosphate and 150 mM NaCl, pH 7.2) at the final concentration of 10 μ M and incubated at 37 °C for 30 min before cooling down to 4 °C. The CD spectra of each sample were acquired on a Jasco spectropolarimeter (Model J-815, Jasco Inc., Japan) at 4 °C using a 5 nm bandwidth, 0.1 nm resolution, 0.1 cm path length, and an average time of 5.0 sec. Spectra were corrected by the subtraction of a blank corresponding to the solvent composition of each sample. Thermal midpoint analysis was used to determine the temperature at which 50% of the 6-HB formed by the CHR and NHR would decompose. It was monitored at 222 nm from 4 °C to 98 °C by applying a thermal gradient of 5 °C/min. The melting curve was smoothed, and the midpoint of the thermal unfolding transition (Tm) values was calculated using Jasco software utilities as described above.
Inhibition of gp41 six-helix bundle formation by sandwich ELISA. Inhibition of gp41 six-helix bundle formation by a testing peptide was determined with a sandwich ELISA described previously 57 . Briefly, a testing peptide (ADS-J1 as a control) at graded concentrations was preincubated with peptide N36 (1 μ M) at 37 °C for 30 min, followed by the addition of peptide C34 (1 μ M) and incubation at 37 °C for another 30 min. The mixture was added to a 96-well polystyrene plate (Costar, Corning Inc., Corning, NY) precoated with anti-N36/C34 antibodies (2 μ g/ml) purified from mouse antisera specifically against the gp41 six-helix bundle 58 . Then, mAb NC-1, HRP-labeled rabbit-anti-mouse IgG (Sigma), and TMB were added in order. A450 was determined by an ELISA reader (Ultra 384, Tecan).
Inhibition activities of AP1, AP2, and AP3 on HIV-1 infection were determined as previously described 57 . For inhibition of HIV-1 IIIB (subtype B, X4) infection,100 TCID 50 of the virus was added to 1 × 10 5 /ml MT-2 cells in RPMI 1640 medium containing 10% FBS in the presence or absence of the test peptide overnight. Then, the culture supernatants were changed to fresh media. On the fourth day post-infection, culture supernatants were collected for detection of p24 antigen by ELISA. For inhibition of infection by the HIV-1 strain Bal (subtype B, R5), M7 cells (1 × 10 5 /ml) were precultured overnight and infected with Bal at 100 TCID 50 in the presence or absence of the test peptide or protein overnight. Then, the culture supernatants were changed to fresh media. On the fourth day post-infection, the culture supernatants were discarded, and fresh media were complemented again. The supernatants were collected on the seventh day post-infection and tested for p24 antigen by ELISA as previously described 55 . The percent inhibition of p24 production was calculated. Analysis of the half-life of peptide inhibitors. Four male SD rats weighing approximately 200 g each were obtained from the Shanghai Medical School Animal Center and were used for the half-life assay. Animals were treated in accordance with the Animal Welfare Act and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication 86-23, revised 1985). Either AP2 or AP3 was intravenously injected at the concentration of 1 mg/ml. After injection, blood samples were acquired from rat orbit at several time points (8 and 30 min and 1.5, 3, 6, 9, 12, and 24 h after peptide injection) and placed in clean tubes. To study the pharmacokinetics of AP2 and AP3 in rats and provide experimental evidence for the possible pharmacokinetics in human, a double-antibody sandwich ELISA method was established for rapid determination of AP2 and AP3 in rat plasma. Briefly, 96-well polystyrene plates (Costar, Corning Inc., Corning, NY) were precoated with antibody against AP2 or AP3 (5 μ g/ml) purified from rabbit anti-sera 59 . They were then preincubated with serum samples diluted 20 times at 37 °C for 1 h, followed by the addition of anti-AP2 or anti-AP3 antibody (1:1000) purified from mouse antisera specifically against AP2 or AP3 59 at 37 °C for another 1 h. Then, HRP-labeled rabbit-anti-mouse IgG (Sigma, USA) and TMB were added in order. Absorbance at 450 nm was determined by an ELISA reader (Ultra 384, Tecan). The standard peptide parameters were obtained first. Then, the plasma peptide concentrations were determined as a function of time, and the half-life was calculated by using PK Solver for Microsoft Excel to obtain pharmacokinetic parameters.
Assessment of sensitivity of peptides to proteolytic digestion by proteinase K and proteolytic enzymes in liver homogenate. The peptides (10 μ g/mL) were prepared in PBS pH 7.2 containing 20 ng/ml proteinase K. The resulting mixture were incubated at 37 °C in a water bath and taken out at different time intervals (0, 5, 15, 30, 60, 120 minutes), followed by quenching the samples with ethyl alcohol and quantitating the peptides by LC-MS analysis as described above.
To test the sensitivity of peptides to the proteolytic enzymes in liver homogenate, 3 male SD rats (250 ± 20 g) were sacrificed under anesthesia. The whole liver was quickly removed from each rat, washed in ice-cold PBS (50 mM, pH 7.2), weighed and cut into small pieces, which were resuspended in PBS to 100 mg wet liver tissue/2.5 ml PBS. The samples were pooled and homogenized, followed by centrifugation at 9,000 g for 20 min at 4 °C. The supernatants were collected. The test peptides were added to the liver homogenate at a final concentration of 10 μ g/ml. The resulting mixture was incubated 37 °C in a water bath, and the residue peptides in the mixture were quantitated as described above. | What is Enfuvirtide? | {
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"text": [
"HIV fusion inhibitor"
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