The DISC1 (disrupted in sсhizophrenia 1) gene is associated with brain dysfunctions, which are involvedin a variety of mental disorders, such as schizophrenia, depression and bipolar disorder. This is the first study toexamine the immune parameters in Disc1-Q31L mice with a point mutation in the second exon of the DISC1 genecompared to mice of the C57BL/6NCrl strain . A flow cytometry assay has shown that intact Disc1-Q31L mice differ from the WT strain by an increase in the percentage of CD3+ T cells, CD3+CD4+ Т helper cellsand CD3+CD4+CD25+ T regulatory cells and a decrease in CD3+CD8+ T cytotoxic/suppressor cells in the peripheralblood. A multiplex analysis revealed differences in the content of cytokines in the brain structures of Disc1-Q31Lmice compared to WT mice. The content of pro-inflammatory cytokines was increased in the frontal cortex  and striatum (IFNγ), and decreased in the hippocampus and hypothalamus. At the same time, thelevels of IL-1β were decreased in all structures being examined. In addition, the content of anti-inflammatory cytokinesIL-4 was increased in the frontal cortex, while IL-10 amount was decreased in the hippocampus. Immuneresponse to sheep red blood cells analyzed by the number of antibody-forming cells in the spleen was higher inDisc1-Q31L mice at the peak of the reaction than in WT mice. Thus, Disc1-Q31L mice are characterized by changes inthe pattern of cytokines in the brain structures, an amplification of the peripheral T-cell link with an increase in thecontent of the subpopulations of CD3+CD4+ T helpers and CD3+CD4+CD25+ T regulatory cells, as well as elevatedimmune reactivity to antigen in the spleen. Animal models have provided valuableopportunities to study the impact of immune dysfunctions andrelated alterations in neurotransmitter and hormonal systemsin the pathogenesis of neuropsychiatric disorders caused bymultiple risk factors, including genetic background. As shownpreviously, animals with genetic predisposition to depressiveor aggressive behavior are characterized by changes in thedistribution and ratio of the main subpopulations of T-cells inthe blood and spleen, immune responsiveness to T-dependentantigen, as well as cytokine variations in the periphery andbrain structures .It is now well established that a variety of social, environmentaland genetic factors may cause inflammatory responses that,over time, may result in development of multiple diseases,including neuropsychiatric disorders . The inflammatory processesare closely associated with alterations in the productionof cytokines , the compositionof T-cell subsets with different functional activities . Analysis of emotional,social and cognitive behaviors of this mice line showeda range of behavioral abnormalities that may be considered asa depression-like endophenotype . TheQ31L mutation in DISC1 gene is also known to be associatedwith changes in the dopaminergic (DA) activity  and other neuromediator systems, which are involvedin the neurobiological mechanisms of psychiatric disordersand in the control of immune function .Disrupted-in-Schizophrenia-1 (DISC1) gene has beenfunctionally linked to brain dysfunctions associated with impairedneurodevelopment processes and intracellular signalingpathways that predispose to schizophrenia, major depression,and bipolar disorder . Several mouse models based on DISC1 dysfunctionhave been generated to date, including a homozygousDisc1-Q31L–/– mice remain to be elucidated. Given a roleof the immune system both in the development of differentpsychoemotional states and in neuroimmunomodulation was also determined.2019), the aim of this study was to analyze the basal contentof T- and B-cells in the peripheral blood and spleen, as wellas the level of pro- and anti-inflammatory cytokines in thebrain structures of Disc1-Q31LAnimals. The experiments were performed in adult (3.0–3.5 months old) homozygous male mice of the Disc1-Q31L–/–strain (n = 23) and there wild type (WT) littermates of theC57BL/6NCrl strain (n = 23) weighing 27–30 g. Mice werebred in the animals facility of the Scientific Research Instituteof Physiology and Basic Medicine .Mice were kept in standard cages  in groups for 5 animals per cage under standardvivarium conditions and free access to food and water.All experimental procedures were performed in accordancewith the requirements of the European Community Directive(86/609/EC) and approved by Local Ethical Committeeof the Scientific Research Institute of Physiology and BasicMedicine, protocol No. 10 (17.12.2015).Design of experiments. The levels of T- and B-lymphocytesand their subpopulations in the peripheral blood andspleen, as well as the content of proinflammatory  and anti-inflammatory (IL-4and IL-10) cytokines in the brain structures  were assessed in intactmice of the Disc1-Q31L and WT strains . The immune reactivity to sheep red blood cells (SRBC)was analyzed by measuring the number of antibody-formingcells (AFC) in the spleen of mice of both strains (n = 13 ofeach strain). SRBC were suspended in saline and injectedonce, intravenously into the tail vein at a dose of 5 · 108.Blood was immediately collected after the animals weredecapitated into tubes containing K3EDTA . Spleens were removed on ice on day 4 afterSRBC injection and placed in tubes with cooled RPMI-1640medium . Brain structures were dissectedon ice; brain samples were frozen in liquid nitrogenand stored at –70 °C until analysis.Determination of cell subpopulation. To analyze cellsubsets, 25 μl of blood was incubated for 30 minutes ina dark place with 1.5 μl (0.2–0.5 μg/μl) labeled rat anti-mousemonoclonal antibodies (MoAB) against surface markers:СD3 , СD4 , СD8 , СD25 (BrilliantViolet 421), СD19  . Erythrocytes of theblood were lysed with Lysing Solution BD FASC . After a 10-minute incubation, the cells werewashed once with phosphate buffered saline (PBS), the cellpellet was resuspended in 100 μl of PBS.6/100 μl of the suspension and placed intoplates in a volume of 100 μl in each well. The cell suspensionwas incubated with the same MoAB as the blood cellsfor 20 minutes, and fixed by adding 1 % paraformaldehyde toeach tube. Isotypic antibodies were used as a control.The spleen was cut into several pieces, and then disaggregatedmechanically into single-cell suspension, which waspassed through a 50 μm cell strainer. The suspension waswashed twice with RPMI-1640 medium at 200 g for 5 minutes.The cell pellet was resuspended in RPMI-1640 medium,adjusted to 1 · 10+ T-lymphocytes, CD3+CD4+T-helpers, CD3+CD8+ cytotoxic/suppressor T-lymphocytes,CD3+CD4+CD25+ T-regulatory cells, CD19+ B-lymphocytesas a percentage of the total number of cells were determined.Immunoregulatory index was measured as a ratio of the contentof CD4+ to CD8+ Т-cells.The study of cell populations was performed on a FACSCANTO™ II flow cytometer  usingmulti-stage gating. At least 50 000 cells were analyzed in eachsample. Data analysis was performed using the FACSDivasoftware. The contents of CD3Determination of cytokines in the brain structures.For the analysis of cytokines, detergent-soluble fractions ofbrain tissues were prepared. The samples were thawed on ice,homogenized in lysis buffer cooled to +4 °C containing PBS(pH 7.4), 0.1 % Triton X-100, 1 mM EDTA, and 1 mM PMSFusing plastic pestles. The homogenates were incubated on icefor 30–40 minutes. The tissue extracts were centrifuged (Centrifuge5415 R) at 4500 rpm for 20 minutes at +4 °C. Cytokineconcentrations were determined in the supernatants. Theconcentration was normalized to tissue weight (pg/g tissue)The content of cytokines in brain homogenates was determinedaccording to the manufacturer’s protocol by multipleximmunoassay on a multiplex protein and nucleic acid analyzer using a kit . The resultswere analyzed using the xPONENT and Analist software.Determination of antibody-forming cells. The immuneresponse was assessed by the relative (per 106 spleen cells)and absolute  numberof IgM-AFC using the standard method .Statistical analysis. The data were analyzed using Statistica10.0 software. To verify whether data were normallydistributed, the Kolmogorov–Smirnov and Shapiro–Wilktests were used. Normally distributed data  were assessedby one-way ANOVA. The Mann–Whitney test was used forabnormally distributed data (cytokine content and AFC number).Data are presented as mean and mean error (M ± m) withsignificance set at a level of p < 0.05.Content of T-cells, their subpopulations, and B-cells in theperipheral blood and spleen of Disc1-Q31L mice. Therewere differences in the content of all analyzed immunocompetentblood cells between nonimmunized Disc1-Q31L andWT mice. The percentage of CD3+ T-lymphocytes in mice of the Disc1-Q31L strain was significantly higher than inWT mice (F(1.18) = 45.2, p < 0.001). Analysis of T-lymphocytesubpopulations showed an increase in the content ofCD3+CD4+ Т-helpers (F(1.17) = 15.5, p < 0.01) in Disc1-Q31Lmice compared to WT strain, while the number of CD3+CD8+T-cytotoxic/suppressor cells was decreased (F(1.17) = 12.6,p < 0.01). As a result, the immunoregulatory index, determinedas the ratio of the content of CD4+ to CD8+ T-lymphocytes, inmutant mice was 1.3 times higher (F(1.18) = 27.5, p < 0.01)than in WT mice. The content of T-regulatory cells with theCD3+CD4+CD25+ phenotype in Disc1-Q31L mice was alsohigher than in WT mice (F(1.17) = 5.3, p < 0.05). The numberof CD19+ B-lymphocytes was decreased in the peripheralblood of mutant mice compared to WT mice (F(1.17) = 5.7,p < 0.05) (see the Table).Cytokines in the brain structures in mice of the Disc1-Q31L strain. Analysis of the cytokine profile in brain structuresof intact Disc1-Q31L mice revealed regional differencesin the content of cytokines between mutant and WT mice.In the frontal cortex, levels of the three pro-inflammatorycytokines IL-6 ( p < 0.01), IL-17 ( p < 0.01) and IFNγ( p < 0.01) were higher in Disc1-Q31L mice than in WT mice,while the level of IL-1β ( p < 0.05) decreased. IL-2 and TNFαlevels were similar between the mutant and WT strains( p < 0.05). As to the content of anti-inflammatory cytokines,the level of IL-4 in Disc1-Q31L mice was higher than inWT mice ( p < 0.01), while the level of IL-10 did not change( p > 0.05) .In the striatum, the content of IFNγ ( p < 0.01) was foundto be increased in Disc1-Q31L mice compared to WT animals.The levels of other pro-inflammatory cytokines – IL-1β( p < 0.01), IL-2 ( p < 0.001) in the mutant mice were lowerthan in WT mice, while the levels of IL-6, IL-17 and TNFαremained unchanged ( p > 0.05). Similarly, there were nosignificant strain differences in the levels of anti-inflammatorycytokines IL-4 and IL-10 ( p > 0.05) .When compared to WT mice, Disc1-Q31L mice showedlower levels of IL-1β ( p < 0.01), IL-2 (p < 0.01) and IL-17( p < 0.05) in the hypothalamus, while the levels of the restcytokines were unchanged ( p > 0.05) .The levels of proinflammatory cytokines IL-1β, IL-2,IL-17, TNFα ( p < 0.05) were significantly lower in the hippocampusof Disc1-Q31L mice than in WT mice, with morepronounced decrease in IFNγ content ( p < 0.001). The levelsof IL-6 were equivalent between Disc1-Q31 and WT mice( p > 0.05). The content of anti-inflammatory cytokine IL-10in the hippocampus of Disc1-Q31 mice was also decreased ( p < 0.05), while the level of IL-4 did not differ from that ofWT mice ( p > 0.05) .Immune reaction of Disc1-Q31L mice to the antigen.Immunization of Disc1-Q31L mice with SRBC produced anincrease of the immune response at the peak of its developmentin the spleen of WT mice. The relative ( p < 0.001) andabsolute ( p < 0.001) numbers of AFC in Disc1-Q31L micewere significantly higher than in WT mice .+ T-helpers, and the ratio of CD4+to CD8+ T-cells . Aggressive behavior, asobserved in a variety of animal models, is also associated withan increase in T-helpers and the CD4+/CD8+ ratio, as well asa higher immune response generated by an antigen .Changes in DISC1 protein activities caused by mutations inthe DISC1 gene are known to be involved in multiple mentaldisorders, such as schizophrenia, depression, bipolar disorder. Alterations in immune variables associated withthese disorders may differentially contribute to disease development.Schizophrenia has been found to be accompanied byelevated serum numbers of B-cells, along with a decrease inthe content of T-cells, CD4+CD8+ T-suppressor/cytotoxic cells, adecrease in the immunoregulatory index and the immuneresponse suppression .On the other hand, depression is characterized by increasingnumbers of CD3+ T-lymphocytes, and their subpopulations, such asCD3+CD4+ T-helpers and CD3+CD4+CD25+ T-regulatorycells, with a consequent increase of the immunoregulatoryindex. At the same time, the mutant mice showed lower percentageof CD3+ T-lymphocytes in the spleen, that might leadto a predominance of CD19+ B-cells, thereby suggesting aredistribution of these cell subsets within the immune system.The present study demonstrates that, compared to WT mice,intact mice of the Disc1-Q31L strain have raised blood levelsof CD3Redistribution of T- and B-lymphocytes, which are knownto produce specific sets of cytokines, and the ratio of thesecells in the immunocompetent organs may significantly affectinflammatory and immune processes characteristic of geneticallydetermined behaviors and psychopathology . It seems, thus, possiblethat higher ability of Disc1-Q31L mice to respond to antigenchallenge, as measured by the numbers of AFC in the spleen,may be related to changes in immune cell distribution amongdifferent compartments of the immune system.Our results have also shown that the pattern of cytokinevariations over brain structures differ in Disc1-Q31L andWT mice and depends on the brain area, in which thesecytokines are localized. The levels of IL-6, IL-17 and IFNγwere found to be simultaneously increased only in the frontalcortex of Disc1-Q31L mice compared to WT animals. Thesepro-inflammatory cytokines has long been known as verypotent signaling molecules of neuroinflammation implicatedin the pathophysiology of depression, bipolar disorder, andschizophrenia .Moreover, the frontal cortex has also been associated withthe development of various psychiatric diseases .Only the IFNγ level was increased in the striatum of Disc1-Q31L mice compared to WT mice, whereas the concentrationsof other cytokines decreased. Levels of pro-inflammatory cytokineswere also lower in the hippocampus and hypothalamusof mutant mice than in WT mice. Changes in the distributionof brain cytokines found in Disc1-Q31L mice suggest thatthis mutation may contribute to neuroinflammation, whichis an important etiological factor for affective disorders. Theobserved increase in the level of anti-inflammatory cytokineIL-4 in the frontal cortex of Disc1-Q31L mice could reflecta compensatory response to the elevations of pro-inflammatorycytokines that occurred in this brain area. These findings areconsistent with previous reports, showing that various formsof depression-like behavior or aggression are associatedwith impaired balance between pro- and anti-inflammatorycytokines in a number of brain regions including the frontalcortex and hippocampus .DISC1 has been found to form a complex with other intranucleartranscription factors, which mediate the expression ofseveral genes implicated in behavioral changes resembling human psychiatric disorders . A wide rangeof studies on behavioral phenotype of Disc1-Q31L producedconflicting results. Some data indicate that mice with Q31Lmutation in Disc1 have a depressive-like enodophenotype, while others did not show significant behavioral differencesin this strain compared with the WT control in any of thetests . Recent evidence suggests that Disc1-Q31L mice may also display aggressive behavior . In contrast to the data obtained in other models, inwhich animals developing depression-like responses showedimmunosuppression , our results revealed higher immunereactivity in Disc1-Q31L mice compared to WT control. Atthe same time, immune parameters characteristic of Disc1-Q31L mice are more relevant to those observed in aggressiveanimals. It may be due to the diverse behavioral phenotype ofthese mice displaying not only depression, but also aggressivebehavior  that is associated with increasedimmune function and specific pattern of cytokines.However, the mechanisms underlying alterations in peripheralimmune parameters and the profile of brain cytokines areunknown. There is growing evidence that immune mediatorssuch as cytokines are involved in the interactions betweenthe immune and neuroendocrine systems and can change theactivity of central neuromediator systems that contribute tocognitive, behavioral, and brain structure abnormalities seenin affective disorders . It is possible that the immune status of Disc1-Q31Lmice could be related to the neurochemical pattern of thebrain characteristic of this strain. Disc1-Q31L mice have beenshown to have decreased levels of DA combined with DOPACincrease in the nucleus accumbens , knownto implicate in neuroimmunomodulation . There is also data thatthe DOPAC/DA ratio, which may reflect the metabolic rate ofDA and synaptic activity, increases under immunostimulationobserved in animals experienced excessive aggression associatedwith elevated activity of the DA system . Taking into account changing DAergicactivity in the brain structures of Disc1-Q31 mice  and the critical role of this system in the control ofaggression and neuroimmunomodulation , it is likely that DA maycontribute to the enhancement of immune function found inthe Disc1-Q31 strain.However, it remains unclear whether variations of centralcytokines are related to brain alterations of monoamines specificfor Disc1-Q31L mice or the Q31L mutation determinestheir profile. Moreover, it has been found that not only neurotransmitterscan affect the production of cytokines , but also cytokines can modulate mediator neurotransmissionand promote changes in the neurochemicalpattern of the brain .+CD4+CD25+ T-regulatory cell subpopulations, as wellas elevated immune reactivity in the spleen induced by theantigen. Alterations in the peripheral immune variables areaccompanied with changes in the distribution of pro- andanti-inflammatory cytokines within brain structures, whichare involved both in the control of different forms of behaviorand in immune function. The Disc1-Q31L mouse strain is apromising model for further study of the relationships betweengenetic factors and neuroimmunological mechanismsand their implication in the development of psychoemotionaldisorders.Our data indicate that the Q31L point mutation in the DISC1gene leading to the substitution of glutamine to leucineat amino acid 31 has a significant influence on immunity and may result in an amplification of peripheral T-cell linkwith an increase in the content of CD3+CD4+ T-helpers andCD3The authors declare no conflict of interest.Al’perina E.L. Involvement of the dopaminergic system in the mechanismsof immunomodulation. 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The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta  emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. Here, we compared B.1.617 variants for neutralization resistance by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. B.1.617.1, B.1.617.2, and AY.1 pseudoviruses showed a modest 1.5- to 4.4-fold reduction in neutralization by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for C.37, P.1, R.1, and B.1.526 pseudoviruses, but 7- and 16-fold reductions for vaccine-elicited and convalescent sera, respectively, were seen for B.1.351 pseudoviruses. Among twenty-three therapeutic antibodies tested, four antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses and six antibodies showed either complete or partial loss of neutralization against B.1.617.1 and AY.1 pseudoviruses. Our results indicate that the current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants. Finally, the P681R substitution confers efficient cleavage of B.1.617 variants’ spike proteins and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants. SARS-CoV-2 trimeric spike (S) glycoprotein on the virion surface binds the angiotensin-converting enzyme (ACE2) to facilitate cellular entry and is the target of therapeutic neutralizing antibodies and vaccines [685|S686 into S1 and S2 subunits, which facilitates subsequent cleavage at the S2′ site (R815|S816) by TMPRSS2 for viral entry into respiratory cells. The S1 subunit spans the N-terminal domain (NTD) and the receptor-binding domain (RBD) within the C-terminal domain (CTD) whereas the S2 subunit spans the fusion peptide and a linker region flanked by heptad repeat regions that drive virus-cell membrane fusion [Since its origin in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally to cause a coronavirus disease 2019 (COVID-19) pandemic that recorded more than 263 million infections and has claimed 5.2 million lives thus far  ,9,10,11.Several key substitutions in the RBD of spike were demonstrated to either enhance affinity towards ACE2 or contribute to immune escape. The E484K substitution in the RBD of B.1.351, P.1, R.1, and B.1.526 variants was previously identified among in vitro escape mutants selected against single antibody and antibody cocktails ,14. SeveThe spike protein of the B.1.617.2 variant contains nine substitutions and deletions compared to the early D614G variant used here as wild type (WT or D614G). The three substitutions  and two deletions  in NTD occur in the NTD antigenic supersite spanning between residues 14–20, 140–158, and 245–264 . In addiThe global dominance and ongoing evolution of B.1.617 lineage variants require continuing assessment of the neutralization potency of convalescent sera, vaccine-elicited sera, and therapeutic neutralizing antibodies against emerging B.1.617 variants. Here, we measured the neutralization potency of convalescent sera, vaccine-elicited sera, and therapeutic neutralizing antibodies against two independent variants each in the Kappa (B.1.617.1) and Delta  lineages and assessed the contribution of the RBD substitutions in conferring resistance. We found that resistance to convalescent and vaccine-elicited sera was predominantly conferred by RBD substitutions E484Q, T478K, and L452R. Furthermore, out of 23 therapeutic neutralizing antibodies tested, Kappa and Delta pseudoviruses displayed complete resistance to five neutralizing antibodies and partial resistance to one antibody. Finally, we showed that the P681R substitution confers enhanced furin processing in spike protein of B.1.617 lineage variants that corresponded to enhanced cell-cell fusion activity. However, only Delta spike protein exhibited greater sensitivity to soluble ACE2 inhibition, implying enhanced ACE2 affinity. This feature along with enhanced cell-cell fusion activity may contribute to the dominance of the B.1.617.2 variant.Use of de-identified sera in this study was approved by the U.S. Food and Drug Administration Research in Human Subjects Committee. Vaccine-elicited sera were collected at the U.S. Food and Drug Administration with written consent under an approved Institutional Review Board (IRB) protocol (FDA IRB Study # 2021-CBER-045).Codon-optimized full-length open reading frames of the S genes of SARS-CoV-2 variants were cloned into pcDNA3.1(+) or pVRC8400 by GenScript . The codon optimization for gene expression in human cell was performed by GenScript’s OptimumGene algorithm system to optimize the following parameters: codon usage bias, GC content, CpG dinucleotides content, mRNA secondary structure, internal chi sites and ribosomal binding sites, negative CpG islands, cryptic splicing sites, premature PolyA sites, PolyT sites, RNA instability motif (ARE), and repeat sequences . The codon-optimized sequence of Wuhan-Hu-1 S gene is shown in n = 10) collected 6–61 days after symptom onset were purchased from Bocabiolistics . Donors were 18–73 years old with six males/four females. The information about the convalescent sera is shown in n = 15) or Moderna mRNA-1273 vaccinated individuals (n = 14) obtained two weeks after the second vaccination were used in this study. Vaccinated individual donors were 21–65 years old with six males/nine females for Pfizer BNT162b2 vaccination and eight males/six females for Moderna mRNA-1273 vaccination. All sera were tested negative for non-specific neutralization using amphotropic murine leukemia enveloped pseudovirus. Vaccinated donors were prescreened for absence of both history of SARS-CoV-2 infection and SARS-CoV-2 neutralizing antibodies prior to vaccination. Twenty-three therapeutic neutralizing antibodies against SARS-CoV-2 spike protein were donated by different pharmaceutical companies for the U.S. government COVID-19 response Therapeutics Research Team efforts to define neutralization profiles against existing and emerging SARS-CoV-2 variants [Convalescent sera from SARS-CoV-2-infected donors  as described previously .6 relative luminescence units (RLU)/mL of luciferase activity were incubated with serially diluted sera or antibodies for two h at 37 °C. Pseudovirus and serum or antibody mixtures (100 μL) were then inoculated onto the plates pre-seeded one day earlier with 3.0 × 104 cells/well. Pseudovirus infectivity was scored 48 h later for luciferase activity. The antibody concentration or inverse of the sera dilutions causing a 50% reduction of RLU compared to control was reported as the neutralization titer. Titers were calculated using a nonlinear regression curve fit . The mean titer from at least two independent experiments each with intra-assay duplicates was reported as the final titer. WT(D614G) pseudovirus was run as a control for every assay.HIV-based lentiviral pseudoviruses with spike proteins were generated as previously described . The B.16 RLU/mL) for one hour at 37 °C and 100 μL of pseudovirus and soluble ACE2 mixture was added to 293T-ACE2.TMPRSS2 cells. Luciferase activity was measured 48 h later. The soluble ACE2 concentration causing a 50% reduction of RLU compared to control was reported as the 50% inhibitory concentration or IC50.For ACE2 neutralization assay, serially diluted recombinant human soluble ACE2 was incubated with indicated pseudovirus .4 cells per well on a 96-well plate. The cells were co-cultivated for 24 h at 37 °C. The culture supernatants were then removed, and cell-cell fusion was scored by determination of the β-gal activity in co-cultured cell lysates using a Galacto-Star kit  according to the manufacturer’s instructions.For measuring spike-protein-mediated cell-cell fusion, β-gal complementation assay was performed as described previously . BrieflyTo ensure equivalent amount of spike cell surface expression levels among treatments, spike-transfected β-gal ω subunit-expressing 293T cells were quantified for cell surface spike levels by flow cytometry. Spike-transfected 293T cells employed in cell-cell fusion assay were concurrently stained with SARS-CoV-2 positive human polyclonal sera at 1:20 dilution, washed twice, and then incubated with FITC-conjugated goat anti-human . The cells were washed twice and then fixed with 2% paraformaldehyde. The results were acquired using BD LSRFortessa™ X-20 Cell Analyzer . The mean fluorescence intensities of spike positive cells were recorded.https://github.com/acorg/Racmacs, accessed on: 15 October 2021) [We created a geometric interpretation of neutralization titers against the tested SARS-CoV-2 pseudoviruses using Racmacs antigenic cartography software (Sam Wilks (2021), Racmacs: R Antigenic Cartography Macros. R package version 1.1.16. er 2021) ,37. The http://www.cbs.dtu.dk/services/ProP/ (accessed on: 15 July 2021) and the PiTou V3 software hosted at http://www.nuolan.net/reference.html (accessed on: 15 July 2021).The prediction of furin-specific cleavage site in spike proteins was computed using the ProP 1.0 Server hosted at p values of less than 0.05 were considered statistically significant. All neutralization titers were log2 transformed for analyses.One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons tests (variants compared to WT(D614G)), Mann–Whitney test for the comparison of two groups with unmatched pairs (Pfizer BNT162b2 compared to Moderna) and geometric mean titers (GMT) with 95% confidence intervals were performed using GraphPad Prism software. The We first investigated the cross-neutralization potency of convalescent sera from individuals infected with SARS-CoV-2 in the U.S. against pseudoviruses bearing spikes of B.1.617.1 and B.1.617.2 variants and their corresponding RBD mutations A. TitersThe C.37 variant also has a substitution at L452 residue (L452Q instead of L452R) along with F490S in the RBD. A modest 1.8-fold reduction in titers against C.37 pseudoviruses was observed compared to WT(D614G) pseudoviruses . A 1.4-fold reduction in titers was observed for pseudoviruses with only the L452Q and F490S substitutions, indicating that these RBD substitutions contribute to C.37 resistance. These findings are in agreement with a prior report showing a 3.3-fold reduction of convalescent sera neutralization titer for C.37 pseudoviruses compared to WT(D614G), as well as L452Q and F490S single substitutions contributing to neutralization resistance . In compBecause the convalescent sera came from individuals who were previously infected by different variants , we alsoSera from the group infected by variants that had the L452R substitution showed similar fold changes in neutralization titers as the group infected by WT(D614G) variants without the L452R except against B.1.617.1 (B) and AY.1 pseudoviruses. Fold changes in neutralization titers against B.1.617.1 (B) and AY.1 variants were 3.3 and 1.3, respectively, for the L452 group, compared to 5.2 and 2.5, respectively, for the WT(D614G) group B,C. The p = 0.1225, Mann–Whitney test). The Moderna mRNA-1273 vaccine-elicited sera trended towards higher neutralization titers against most variants compared to Pfizer/BioNtech BNT162b2, possibly due to higher vaccine mRNA content and greater interval between priming and boosting for Moderna mRNA-1273 (4 weeks vs. 3 weeks for Pfizer/BioNtech BNT162b2) [We next assessed the neutralization potency of mRNA vaccine-elicited sera against WT(D614G) and B.1.617 variant pseudoviruses. Sera from fourteen individuals who received two doses of Moderna mRNA-1273 vaccine and fifteen individuals who received two doses of Pfizer/BioNtech BNT162b2 vaccine were collected approximately two weeks after the second immunization. Each vaccine-elicited serum had high neutralization titers against WT(D614G) pseudoviruses, ranging between 578 and 3935 for Pfizer/BioNtech BNT162b2 and 651 and 5853 for Moderna mRNA-1273 A,B (PfizNT162b2) . CompareNT162b2) ,39.We also investigated the contribution of individual RBD substitutions of B.1.617.1  and B.1.617.2/AY.1  on the D614G background. Titers against L452R (GMT 935 for Pfizer and 1781 for Moderna) and E484Q (GMT 798 for Pfizer and 1429 for Moderna) alone trended slightly lower. Likewise, titers against K417N (GMT 1208 for Pfizer and 2070 for Moderna) and T478K (GMT 1046 for Pfizer and 2113 for Moderna) alone or in L452R + T478K combination (GMT 964 for Pfizer and 1796 for Moderna) remained comparable to GMTs of WT(D614G) .Similarly, GMTs against B.1.1.7 (955 for Pfizer and 1917 for Moderna) and B.1.429 variant with the L452R substitution (1063 for Pfizer and 1799 for Moderna) were comparable to those against WT(D614G). Consistent with previous observations, the B.1.351 variant (GMT 169 for Pfizer and 306 for Moderna) displayed ~7-fold lower titers compared to WT(D614G), whereas C.37, P.1, R.1, and B.1.526 variants displayed modestly reduced titers that are similar to the titers against B.1.617 pseudoviruses (GMT 452–707 for Pfizer and GMT 824–1332 for Moderna). Overall, the trends in neutralization titers for the vaccine-elicited sera against a large panel of variant pseudoviruses were similar to those for convalescent sera, though the GMTs were approximately 3- to 5-fold higher for vaccine-elicited sera.A prior study showed that convalescent sera and vaccine-elicited antibody neutralization titers against pseudoviruses bearing spikes containing L452R-E484Q-P681R substitutions displayed 2–5-fold reduction, compared to the neutralization titers against WT(D614G) pseudoviruses . In thisn = 10,000 antigenic maps were made by resampling sera with replacement  pseudovirus and the WT(D614G) sera and L452R sera, including K417N, L452R, T478K, L452Q + F490S, and B.1.429 (0.25 to 0.72 antigenic units (AU) from WT . B.1.617The antigenic map of the same SARS-CoV-2 variant pseudoviruses for vaccine-elicited sera showed similar patterns to the convalescent sera map but with some notable differences. Again, pseudoviruses K417N, L452R, T478K, L452Q + F490S, and B.1.429 were close to WT(D614G) (0.05–0.51 AU). However, E484Q and C.37 were both closer to WT(D614G) but were poorly coordinated and extended in elongated shapes around WT(D614G). B.1.617.1 was also slightly closer to WT(D614G) at 1.55 AU and was positioned adjacent to the other variants. Unexpectedly, AY.1 was between B.1.617.2 and B.1.351 and was further away from WT(D614G) (1.89 AU) than in the convalescent sera map (1.60 AU), being positioned only slightly closer to WT(D614G) than B.1.351 was from WT(D614G) (3.02 AU). These antigenic maps reinforce what was observed in terms of neutralizing antibody titers. Compared to the convalescent sera map, there was a larger antigenic difference between AY.1 and WT(D614G) on the vaccine-elicited sera map.Notably, given that the vaccine-elicited sera had much higher titers than convalescent sera across variants, the titers against AY.1 were still higher in vaccinated individuals, meaning the antigenic difference may not translate into loss of vaccine protection. Furthermore, vaccine-elicited sera saw a smaller difference between B.1.351 and WT(D614G) than the convalescent sera, which may have aligned AY.1 and B.1.351 closer together. Overall, while these antigenic maps provide meaningful information on the relative positions of antigens, they are limited by the sera being so tightly clustered. Future antigenic maps with sera against distinct variants would enable more accurate evaluation of antigenic variation among the variants.We next evaluated 23 clinical-stage therapeutic neutralizing antibodies for potency against the B.1.617 variants. These antibodies were evaluated as part of the U.S. Government COVID-19 response effort to inform the clinical testing and use of these antibodies [B.1.617.1 pseudoviruses displayed complete resistance (>50-fold) to five nAbs  and partial resistance (10–50-fold) to one nAb (H) . The E48B.1.617.2 pseudoviruses displayed complete resistance (>50-fold) to three nAbs  and partial resistance (10–50-fold) to one nAb (H) B. The L4Prior reports indicated increased infectivity of pseudoviruses containing the L452R substitution in spike in 293T-ACE2.TMPRSS2 cells due to L452R conferring enhanced RBD affinity to ACE2 ,27. We t50) of sACE2 against B.1.617.2  was 4.2-fold lower, compared to WT(D614G) (IC50: 2.88 µg/mL)  and L452R + T478K  displayed 2.4- and 2.3-fold higher sensitivity to inhibition by sACE2. However, pseudoviruses with only spike RBD substitutions , as well as the B.1.617.1 (IC50:2.03 µg/mL) and AY.1 (IC50:1.97 µg/mL) spikes, displayed comparable IC50 to WT(D614G) (0.5–1.5-fold change)  . In agre change) . These fSince P681H enhanced proteolytic processing of B.1.1.7 spike ,49, we nTo gain further insight into the furin processing efficiency at the S1/S2 site of the B.1.617 S, we undertook a bioinformatic approach utilizing the PiTou and ProP furin cleavage prediction tools, comparing B.1.617 to the Wuhan-Hu-1 (D614) prototype spike and B.1.1.7 spike, as well as spike proteins of several lineage-specific mammalian and animal CoVs. The PiTou algorithm combines a hidden Markov model and knowledge-based cumulative probability score functions for the functional characterization of a 20 amino acid cleavage motif from P14 to P6′ for furin binding and cleavage, whereas ProP predicts furin cleavage sites based on experimental data-derived networks ,54. Both679- R685 and E661- R685 have been reported to have host neuropilin-1 attachment [While SARS-CoV-2 S1/S2 P−R−R−A−R furin cleavage site conforms to a minimal furin recognition motif, R−X−X−R, the presence of H/R instead of P increases the total number of basic residues to four. This presence of basic residue H/R results in additional electrostatic and intramolecular hydrogen bonding to gain substrate turnover . In facttachment  and staptachment .Furin cleavage score analysis of CoV S1/S2 cleavage sites. CoV spike and influenza hemagglutinin (HA) sequences were analyzed using the ProP 1.0 and PiTou 3.0 furin prediction algorithms, generating a score with bold numbers indicating predicted furin cleavage. “|” denotes the position of the furin cleavage site. Sequences corresponding to the S1/S2 region of NL63 (APF29063.1), 229E (ABB90529.1), SARS-CoV-2 (QHD43416.1), SARS-CoV (AAT74874.1), MERS-CoV (AFS88936.1), HCoV-HKU1 (AAT98580.1), HCoV-OC43 (APU51936) Bat-SL-CoVZC45 (AVP78031.1) BatCoV-HKU4 (YP_001039953.1), Influenza A/Chicken/Hong Kong/822.1/01/H5N1 HA (AF509026.2), Influenza A virus HA (A/Wisconsin/67/2005(H3N2) (ACF54576.1), infectious bronchitis virus (QIV13719.1), CoV-HKU9 (YP_001039971), BatCoV-PML (AGY29650), BatCoV-HKU5 (YP_001039962.1), Bat-CoV RaTG13 (QHR63300.2), and Bat-SL-CoVZXC21 (AVP78042.1) were obtained from GenBank. Sequences corresponding to the S1/S2 region of SARS-CoV-2 B.1.1.7 (EPI_ISL_1374509), B.1.617.1 (EPI_ISL_1841346), B.1.617.2 (EPI_ISL_2229775), B.1.617.3 (EPI_ISL_2157058), Bat CoV RmYN02 (EPI_ISL_412977), as well as HA of Influenza A virus ) (EPI1859607) were obtained from GISAID.To quantify whether higher ACE2 binding and furin cleavage of B.1.617.2 spike augments fusion between virus and/or cell membranes, we performed cell-cell fusion assays by complementing β-galactosidase subunits in spike-transfected effector cells and 293T-ACE2.TMPRSS2 target cells. Compared to WT(D614G), both B.1.617.1 and B.1.617.2 spikes induced significantly higher cell-cell fusion activity when controlled for spike cell surface expression (4000 MFI of spike protein on cell surface) B. Our fiHere we show that pseudoviruses bearing B.1.617.1 spike with L452R and E484Q substitutions, and B.1.617.2 spike with K417N, L452R and T478K substitutions, have modestly reduced susceptibility to neutralization by Pfizer/BioNtech BNT162b2 or Moderna mRNA-1273 vaccine-elicited sera and convalescent sera compared to pseudoviruses bearing WT(D614G) spike. The individual L452R, T478K, E484Q, and dual L452R + T478K substitutions accounted for most but not all of the reduction in neutralization potency of the sera, suggesting contributions from substitutions in the NTD/CTD. Neutralization titers, as well as antigenic maps, indicated that the full set of RBD substitutions in combination with substitutions outside the RBD contributes to antigenic differences of B.1.617.1, B.1.617.2, and C.37 variants. Antigenic distances between the variants also tended to be more spaced apart in the map generated by the convalescent sera compared to the vaccine-elicited sera. Limitations in our study include the small number of sera samples in the convalescent and vaccine cohorts. Potential differences in COVID-19 severity in the convalescent sera cohort and time of sera collection could also affect neutralization titers. Nonetheless, most sera from convalesced and vaccinated individuals neutralized the B.1.617.1, B.1.617.2, and AY.1 variants. Furthermore, 17 of 23 therapeutic neutralizing antibodies retained complete neutralization against B.1.617 variants. Resistance to the remaining therapeutic neutralizing antibodies is due to RBD substitutions, K417N, L452R, and E484Q, but not T478K. These findings suggest that the two-dose immunization with current mRNA vaccines will likely induce protective immunity against the tested B.1.617 variants. However, as B.1.617.2 variants continue to evolve, it will be important to continue to monitor how new substitutions in spike impact their resistance to therapeutic neutralizing antibodies and vaccine efficacy.


With an aging population and increasing healthcare utilization, the frequency of hospital-acquired pneumonia (HAP) is expected to increase. Since HAP is life threatening, appropriate diagnosis and treatment are required; however, large-scale Japanese data focusing on patient profiles and treatment patterns is lacking.The demographics and treatment patterns of HAP were examined using a large-scale Japanese claims database from Jan. 2016 to Apr. 2018. The HAP population included patients who received injection antibiotics ≧3 consecutive days after admission, but not within 2 days after admission, and those whose reason for hospitalization was not pneumonia but had a diagnosis of pneumonia after hospitalization (based on ICD-10 codes).2,968 HAP patients  contributing 2,979 total HAP episodes were included. The 12-month pre-index mean Charlson Comorbidity Index (CCI) score was 4.0±3.1 (mean±SD), CCI scores ≧4 comprised 44.0%. Most HAP episodes (77.6%) occurred ≧5 days after hospitalization. During the 12month pre-index period including outpatients, 84.9% of patients had some type of pneumonia record, 9.1% had VAP (ventilator associated pneumonia) records, and 7.4% had anti-MRSA prescription records. For post-index HAP treatment, ampicillin/sulbactam  and piperacillin/tazobactam  were frequently prescribed as the first antibiotic prescription. Ceftriaxone (19.4%) and meropenem (9.8%) were also frequently prescribed. Examinations prescribed during HAP: 30.5% blood culture tests, 28.2% sputum examinations and 29.2% urine antigen tests. The overall mortality rate of HAP in overall hospitalization post-index was 22.0%, in which 14.4% of deaths occurred within 30 days. The mean (±SD) length of overall hospital stay was 49.9 (±34.2) days (11.3 days for HAP period), with 12.4% ICU use and 17.6% ventilator use. The median total cost during hospitalization was ¥1,924,848.18 .The data revealed patient characteristics, treatment patterns, mortality rates and healthcare costs in Japanese HAP patients. This database approach should prove useful for discussing antibiotics usage trends in highly aging Japan.Masahiro Kimata, PhD, MSD K.K., Tokyo, Japan (Employee) Yosuke Aoki, MD, PhD, MSD K.K., Tokyo, Japan SHIONOGI & Co., Ltd  Adachi Noriaki, n/a, MSD K.K., Tokyo, Japan (Employee) Takeshi Akiyama, MSc, MSD K.K., Tokyo, Japan (Independent Contractor) Akiko Harada, n/a, MSD K.K., Tokyo, Japan (Employee)


The phase IIIb ATLAS-2M study demonstrated non-inferiority of long-acting (LA) cabotegravir (CAB) + rilpivirine (RPV) dosed every 8 weeks (Q8W) compared with every 4 weeks (Q4W) for maintenance of virologic suppression. Hepatitis C virus (HCV) co-infection occurs in ~6% of people with HIV due to shared modes of transmission. We report efficacy and safety of CAB + RPV LA in participants with HIV/HCV co-infection in ATLAS-2M.Participants with HIV-1 RNA < 50 c/mL receiving CAB + RPV LA Q4W (transitioned from ATLAS [NCT02951052]) or oral comparator ART were randomized 1:1 to receive CAB + RPV LA Q4W or Q8W. Baseline HCV RNA was assessed by polymerase chain reaction. Participants with symptomatic chronic HCV infection requiring treatment within 12 months or liver enzymes not meeting entry criteria were excluded. Week 48 assessments included proportion with HIV-1 RNA ≥50 and < 50 c/mL , general and hepatic safety, and pharmacokinetics.HIV/HCV co-infection was present in 10 (1%) of 1045 participants, 60% of whom were female at birth. At Week 48, 9/10 (90%) and 972/1035 (94%) participants with HIV/HCV co-infection and HIV mono-infection, respectively, had HIV-1 RNA < 50 c/mL . No participants with HIV/HCV co-infection had HIV-1 RNA ≥50 c/mL (vs 14/1035 [1%] with HIV mono-infection) or confirmed virologic failure through Week 48 (vs 10 [1%] with HIV mono-infection); 1/10 (10%) discontinued for reasons other than adverse events (AEs). Excluding injection site reactions (ISRs), AEs and serious AEs were reported in 4 (40%) and 0 participants with HIV/HCV co-infection, respectively; the only AE reported in >1 participant was injection site pain . In participants with HIV/HCV co-infection, all ISRs were grade 1/2; none led to withdrawal. No hepatic laboratory abnormalities were reported in participants with HIV/HCV co-infection through Week 48; rates were low in those with HIV mono-infection (Table). Plasma CAB and RPV concentrations were similar between groups.CAB + RPV LA was effective and well tolerated in this small cohort of participants with HIV and asymptomatic HCV co-infection.Ronald D’Amico, DO, MSc, GlaxoSmithKline (Shareholder)ViiV Healthcare (Employee) Paul Benn, MB ChB FRCP, ViiV Healthcare (Employee) Shanker Thiagarajah, MB ChB, GlaxoSmithKline  Susan L. Ford, PharmD, GlaxoSmithKline (Shareholder)ViiV Healthcare (Employee) Eileen Birmingham, MD, MPH, Janssen Research and Development  Ojesh R. Upadhyay, MPH, MBA, GlaxoSmithKline (Employee) Louise Garside, PhD, GlaxoSmithKline (Employee) Rodica Van Solingen-Ristea, MD, Janssen Research and Development (Employee)ViiV Healthcare (Employee) Kati Vandermeulen, M.SC., Janssen Research and Development (Employee) William Spreen, PharmD, GlaxoSmithKline (Shareholder)ViiV Healthcare (Employee)


Scientific Reports 10.1038/s41598-021-99672-4, published online 11 October 2021Correction to: The Preprint section in the original version of this Article was omitted. The Preprint section now reads:58.”“This article was submitted to an online preprint archiveAs a result Reference 58 was omitted, which is listed below.et al. Host Genetics Underlying Pathological Outcomes to Mycobacterium Avium Subsp. Paratuberculosis Infection is Governed by Distinct Genetic Variants. Preprint at 10.21203/rs.3.rs-668666/v1 (2021).58. Canive, M., The original Article has been corrected.


This study examined physical activity (FITNESS) and social relationships (FRIENDS) on social engagement among community older adults. Members from two Florida aging-in-village programs participated. Three five-Likert scales were used: A 5-item FITNESS , 4-item FRIEND , and a 3-item social engagement scales  . Among the 96 participants, 79% were females, 91% were whites, 56% were married, 86% had college education, and 46% living alone. Mean age was 70.7 (SD=10.10). Participants reported at least 30-min. physical activity about 4.2 days per week. Overall social engagement was high (mean=4.38), FITNESS was median (mean=3.46), and FRINED was high (mean=4.19). FITNESS was significant to more 30-min. physical activity. Yet, higher FITNESS, FRIENDS, age, and volunteers were all significant to social engagement. Results has implications on promoting social engagement among older adults participating in aging-in-community programs.


Staphylococcus aureus bacteremia (pMRSAB) is unclear. Vancomycin plus ceftaroline (V/C) has demonstrated potent in vitro synergistic activity against MRSA; however, clinical data is limited. Thus, we sought to evaluate V/C salvage therapy for pMRSAB.The preferred antibiotic salvage regimen for persistent methicillin-resistant ≥ 72 hours, received anti-MRSA monotherapy initially, and subsequently received V/C ≥ 24 hours. Patients were excluded if they received other anti-MRSA antibiotics within 72 hours of V/C initiation. The primary outcome was time to BC clearance following V/C initiation. Secondary outcomes included 90-day all-cause mortality, microbiological cure, 90-day MRSAB recurrence, and length of stay (LOS). Microbiological cure was defined as BC clearance.This was a single-center, retrospective cohort study of patients with MRSAB who received V/C salvage therapy between 1/1/2016-3/10/2021. Adult patients were included if blood cultures (BC) were positive for MRSA for Of 178 patients identified, 20 were evaluated after inclusion and exclusion criteria were applied. Mean (SD) age and Pitt Bacteremia score were 38.5 (14.5) years and 4.2 (3.1), respectively. Most patients were male (70%), intravenous drug users (65%), and admitted to the intensive care unit (65%). The most common source was intravenous drug use (55%) and the majority had infective endocarditis (70%). All patients received infectious disease consultation and median (IQR) vancomycin AUC:MIC was 527 . Source control, if possible, was obtained in most patients (55%). Median (IQR) time to bloodstream clearance from first positive BC and from when ceftaroline was initiated was 9.7  and 2.4  days, respectively. 90-day all-cause mortality, microbiological cure, and 90-day MRSAB recurrence occurred in 35%, 95%, and 5% of patients, respectively. Median (IQR) LOS was 25  days.To our knowledge, this is the largest cohort to evaluate V/C for pMRSAB. Patients were medically complex; however, median time to MRSAB clearance following ceftaroline initiation was < 2.5 days and microbiological cure was obtained in nearly all patients. V/C may represent a potential salvage regimen for pMRSAB.Wesley D. Kufel, PharmD, Melinta (Research Grant or Support)Merck (Research Grant or Support)Theratechnologies, Inc. (Advisor or Review Panel member) Jeffrey Steele, Pharm.D., Paratek Pharmaceuticals (Advisor or Review Panel member)


In this study, we characterized blaNDM-carrying plasmids in Enterobacterales isolated in Vietnam, and identified several possible cases of horizontal transfer of plasmids both within and among species of bacteria. Twenty-five carbapenem-nonsusceptible isolates from a medical institution in Hanoi were sequenced on Illumina short-read sequencers, and 13 blaNDM-positive isolates, including isolates of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Morganella morganii, and Proteus mirabilis, were further sequenced on an Oxford Nanopore Technologies long-read sequencer to obtain complete plasmid sequences. Almost identical 73 kb IncFII(pSE11)::IncN hybrid plasmids carrying blaNDM-1 were found in a P. mirabilis isolate and an M. morganii isolate. A 112 kb IncFII(pRSB107)::IncN hybrid plasmid carrying blaNDM-1 in an E. coli isolate had partially identical sequences with a 39 kb IncR plasmid carrying blaNDM-1 and an 88 kb IncFII(pHN7A8)::IncN hybrid plasmid in a C. freundii isolate. 148–149 kb IncFIA(Hl1)::IncA/C2 plasmids and 75–76 kb IncFII(Yp) plasmids, both carrying blaNDM-1 were shared among three sequence type 11 (ST11) isolates and three ST395 isolates of K. pneumoniae, respectively. Most of the plasmids co-carried genes conferring resistance to clinically relevant antimicrobials, including third-generation cephalosporins, aminoglycosides, and fluoroquinolones, in addition to blaNDM-1. These results provide insight into the genetic basis of CRE in Vietnam, and could help control nosocomial infections.Carbapenem-resistant Enterobacterales (CRE) represent a serious threat to public health due to the lack of treatment and high mortality. The rate of antimicrobial resistance of Enterobacterales isolates to major antimicrobials, including carbapenems, is much higher in Vietnam than in Western countries, but the reasons remain unknown due to the lack of genomic epidemiology research. A previous study suggested that carbapenem resistance genes, such as the carbapenemase gene  The same integron cassette was observed on E. coli pHPA  isolated from a human in Japan in 2002 , which had partially identical sequences with two plasmids, pMH17-012N_1  and pMH17-012N_2 , both found in C. freundii MH17-012N (isolated in 2017) (E. coli pMH13-051M_1 [IncFII(pRSB107)::IncN plasmid] and C. freundii pMH17-012N_1  commonly carried blaNDM-1 and blaTEM-1b, and pMH13-051M_1 further carried blaCTX-M-55 and rmtB. C. freundii pMH17-012N_2 [larger IncFII(pHN7A8)::IncN plasmid] carried no known AMR genes, but did have a set of conjugation-associated type IV secretion system (T4SS) genes, which shared identical sequences with E. coli pMH13-051M_1. The genetic structures surrounding blaNDM-1 in E. coli pMH13-051M_1 and C. freundii pMH17-012N_1 contained MGEs, including ISAba125, ISCR21, and two IS26, and shared identical sequences with Tn125 (in 2017) . E. colith Tn125 : 98.6% ablaNDM-1–carrying plasmids identified in Vietnam with those reported in other countries. A set of conjugation-associated T4SS genes in P. mirabilis pMH13-009N_1 [72.6 kb plasmid with IncFII(pSE11)::IncN replicons] was partially identical with Salmonella enterica FDAARGOS_70 plasmid unnamed1  isolated from a human in the United States in 2013, which carried blaTEM-1b (S. enterica FDAARGOS_70 plasmid unnamed1 did not carry blaNDM-1 as P. mirabilis pMH13-009N_1 did. C. freundii pMH17-012N_2 [87.6 kb plasmid with IncFII(pHN7A8)::IncN replicons and no AMR genes] was highly identical with E. coli p103-2-4  from a goose farm in China in 2018 and with S. enterica serovar Enteritidis p12367A  isolated from a human in China in 2013  shared nearly identical blaNDM-1-carrying plasmids, pMH15-289M_1, pMH16-398D_1, and pMH15-258M_1 , and all of these plasmids carried other AMR genes, such as blaCTX-M-15, blaOXA-1, rmtC, and qnrB9 ::IncA/C2 replicons, accession: CP021952, 99.8% identity in 82% region of pMH15-289M_1] in the United States and with E. coli pK71-77-1-NDM  isolated from a human in Norway in 2010. The plasmids tig00000169_pilon and pK71-77-1-NDM had identical sequences in the T4SS region of pMH15-289M_1, and carried blaNDM-1, blaCMY-6, and rmtC, and the plasmid tig00000169_pilon further carried blaSHV-11 and qnrB58. K. pneumoniae MH16-398D had 235 sequence variants , and K. pneumoniae MH15-258M had six sequence variants (five SNVs and one replacement) relative to K. pneumoniae MH15-289M.Furthermore, three nd qnrB9 . blaNDM-K. pneumoniae isolates belonging to ST395  shared nearly identical blaNDM-1-carrying plasmids, pMH15-208H_1, pMH15-191M_1, and pMH13-055M_1 , and all plasmids carried another AMR gene, rmtB  replicon, accession: CP043383, 99.9% identity in 98% region of pMH15-208H_1] isolated from a human in China in 2018 and with K. pneumoniae pSECR18-2374C  isolated from a human in South Korea in 2018. E. hormaechei pNDM1_045001 and K. pneumoniae pSECR18-2374C had a partially identical sequence of conjugation-associated T4SS genes with pMH15-208H_1. E. hormaechei pNDM1_045001 carried blaNDM-1, blaTEM-1b, and rmtB, and K. pneumoniae pSECR18-2374C carried blaNDM-4, blaTEM-1b and rmtB. K. pneumoniae MH15-191M had three sequence variants (two SNVs and one MNV) and K. pneumoniae MH13-055M had only one SNV relative to K. pneumoniae MH15-208H.Another three ne, rmtB . blaNDM-th Tn125 : 99.5–10E. coli, C. freundii pMH17-012N_1 (39.2 kb plasmid with IncR replicon), pMH17-012N_2 [87.6 kb plasmid with IncFII(pHN7A8)::IncN replicons], E. coli pMH13-051M_1 [111.5 kb plasmid with IncFII(pRSB107)::IncN replicons], K. pneumoniae pMH15-258M_1 [148.8 kb plasmid with IncFIA(Hl1)::IncA/C2 replicons], and K. pneumoniae pMH15-208H_1 [75.4 kb plasmid with IncFII(Yp) replicon] were not transferred to recipient E. coli under our experimental conditions though a set of T4SS-associated genes were detected in C. freundii pMH17-012N_2, E. coli pMH13-051M_1, K. pneumoniae pMH15-258M_1, and K. pneumoniae pMH15-208H_1.In bacterial conjugation experiments with K. pneumoniae to third-generation cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones were 66.4%, 17.1%, 29.5%, and 53.0%, respectively, in Vietnam  according to the following protocol. The same amount of Luria-Bertani (LB) broth cultures of each donor bacteria and the recipient azide–resistant E. coli J53 , were mixed and spotted onto Mueller-Hinton agar and then incubated at 37°C overnight. The mixed cells were recovered and suspended into PBS buffer, plated onto LB agar after 10-fold serial dilution, and incubated at 37°C overnight. Transconjugants were selected on LB agar containing 2 μg/mL of meropenem and 100 μg/mL of sodium azide.Bacterial conjugation was performed using six S1 TableAlso, sequence types of multilocus sequence typing (MLST) analysis determined from genomes, carbapenemase genes detected by ResFinder in genomes, sizes and contigs of genomes, coverages in short-read sequencing, Illumina sequencing platforms, and accession numbers of genomes are shown. According to the CLSI 2020 guidelines, Breakpoints of meropenem and imipenem are as follows: ≤1 μg/mL, susceptible; 2 μg/mL, intermediate; ≥4 μg/mL, resistant (R).(TIFF)Click here for additional data file.S2 TableAlso, replicon types detected by PlasmidFinder and antimicrobial resistance genes (ARGs) detected by ResFinder in plasmids, sizes of plasmids, and coverages in long-read sequencing, and accession numbers of plasmids are shown.(TIFF)Click here for additional data file.


It has come to our attention that figures 1 and 3 in this article have been reproduced by the authors without first obtaining permission from the original publishers or authors:Figure 1: Akerfelt M, Morimoto RI, Sistonen L: https://dx.doi.org/10.1038/nrm2938?utm_medium=email&utm_source=transaction Heat shock factors: integrators of cell stress, development and lifespan. Nat Rev Mol Cell Biol. 2010, 11:545-55. https://dx.doi.org/10.1038/nrm2938 10.1038/nrm2938 Figure 3: Pockley AG. https://www.cambridge.org/core/journals/expert-reviews-in-molecular-medicine/article/abs/heat-shock-proteins-in-health-and-disease-therapeutic-targets-or-therapeutic-agents/21095BFDB742E7876E4D1FDBF33E971C Heat shock proteins in health and disease: therapeutic targets or therapeutic agents? Expert Rev Mol Med. 2001, 3:1-21. https://dx.doi.org/10.1017/S1462399401003556 10.1017/S1462399401003556.In addition, Figure 3 was also not properly attributed to the original source. This was later fixed via correction. After confirming with the publisher of Figure 1 that permission to republish was not obtained, and receiving no response from the authors despite multiple attempts, we have made the decision to retract this article and completely remove Figures 1 and 3 from the online and PDF versions of the article.