Sequence amplification via cell passaging creates spurious signals of positive adaptation in influenza virus H3N2 hemagglutinin.
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ABSTRACT:
SUBMITTER: McWhite CD
PROVIDER: S-EPMC5049878 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We have carried out a comprehensive analysis of the determinants of human influenza A H3 hemagglutinin evolution. We consider three distinct predictors of evolutionary variation at individual sites: solvent accessibility (as a proxy for protein fold stability and/or conservation), Immune Epitope Database (IEDB) epitope sites (as a proxy for host immune bias), and proximity to the receptor-binding region (as a proxy for one of the functions of hemagglutinin-to bind sialic acid). Individually, these quantities explain approximately 15% of the variation in site-wise dN/dS. In combination, solvent accessibility and proximity explain 32% of the variation in dN/dS; incorporating IEDB epitope sites into the model adds only an additional 2 percentage points. Thus, while solvent accessibility and proximity perform largely as independent predictors of evolutionary variation, they each overlap with the epitope-sites predictor. Furthermore, we find that the historical H3 epitope sites, which date back to the 1980s and 1990s, only partially overlap with the experimental sites from the IEDB, and display similar overlap in predictive power when combined with solvent accessibility and proximity. We also find that sites with dN/dS > 1, i.e., the sites most likely driving seasonal immune escape, are not correctly predicted by either historical or IEDB epitope sites, but only by proximity to the receptor-binding region. In summary, a simple geometric model of HA evolution outperforms a model based on epitope sites. These results suggest that either the available epitope sites do not accurately represent the true influenza antigenic sites or that host immune bias may be less important for influenza evolution than commonly thought.
| S-EPMC4447415 | biostudies-literature
Project description:Gene-environment interactions have the potential to shed light on biological processes leading to disease and to improve the accuracy of epidemiological risk models. However, relatively few such interactions have yet been confirmed. In part this is because genetic markers such as tag SNPs are usually studied, rather than the causal variants themselves. Previous work has shown that this leads to substantial loss of power and increased sample size when gene and environment are independent. However, dependence between gene and environment can arise in several ways including mediation, pleiotropy, and confounding, and several examples of gene-environment interaction under gene-environment dependence have recently been published. Here we show that under gene-environment dependence, a statistical interaction can be present between a marker and environment even if there is no interaction between the causal variant and the environment. We give simple conditions under which there is no marker-environment interaction and note that they do not hold in general when there is gene-environment dependence. Furthermore, the gene-environment dependence applies to the causal variant and cannot be assessed from marker data. Gene-gene interactions are susceptible to the same problem if two causal variants are in linkage disequilibrium. In addition to existing concerns about mechanistic interpretations, we suggest further caution in reporting interactions for genetic markers.
| S-EPMC4157149 | biostudies-literature
Project description:The hemagglutinin (HA) of influenza A(H3N2) virus responsible for the 1968 influenza pandemic derived from an avian virus. On introduction into humans, its receptor binding properties had changed from a preference for avian receptors (?2,3-linked sialic acid) to a preference for human receptors (?2,6-linked sialic acid). By 2001, the avidity of human H3 viruses for avian receptors had declined, and since then the affinity for human receptors has also decreased significantly. These changes in receptor binding, which correlate with increased difficulties in virus propagation in vitro and in antigenic analysis, have been assessed by virus hemagglutination of erythrocytes from different species and quantified by measuring virus binding to receptor analogs using surface biolayer interferometry. Crystal structures of HA-receptor analog complexes formed with HAs from viruses isolated in 2004 and 2005 reveal significant differences in the conformation of the 220-loop of HA1, relative to the 1968 structure, resulting in altered interactions between the HA and the receptor analog that explain the changes in receptor affinity. Site-specific mutagenesis shows the HA1 Asp-225?Asn substitution to be the key determinant of the decreased receptor binding in viruses circulating since 2005. Our results indicate that the evolution of human influenza A(H3N2) viruses since 1968 has produced a virus with a low propensity to bind human receptor analogs, and this loss of avidity correlates with the marked reduction in A(H3N2) virus disease impact in the last 10 y.
| S-EPMC3535595 | biostudies-literature
Project description:UnlabelledNeutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses.ImportanceAntibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.
| S-EPMC4054433 | biostudies-literature
Project description:H3N2 influenza viruses have now circulated in the human population for 43 years since the pandemic of 1968, accumulating sequence changes in the hemagglutinin (HA) and neuraminidase (NA) that are believed to be predominantly due to selection for escape from antibodies. Examination of mutations that persist and accumulate led to identification of antigenically significant mutations that are contained in five antigenic sites (A-E) mapped on to the H3 HA. In early H3N2 isolates, antigenic site A appeared to be dominant while in the 1990s site B seemed more important. To obtain experimental evidence for dominance of antigenic sites on modern H3 HAs, we have measured antibodies in plasma of human subjects who received the 2006-07 trivalent subunit influenza vaccine (H3 component A/Wisconsin/67/05) or the 2008-09 formulation (H3 component A/Uruguay/716/07). Plasmas were tested against expressed HA of Wisconsin-like influenza A/Oklahoma/309/06 and site-directed mutants in antigenic site A (NNES121-124ITEG, N126T, N133D, TSSS135-138GSNA, K140I, RSNNS142-146PGSG), and antigenic site B (HL156-157KS, KFK158-160GST, NDQI189-192QEQT, A196V). "Native ELISA" analysis and escape mutant selection with two human monoclonal antibodies demonstrated that antibody E05 binds to antigenic site A and 1_C02 binds to site B. We find that most individuals, after vaccination in seasons 2006-07 and/or 2008-09, showed dominance of antigenic site B recognition over antigenic site A. A minority showed dominance of site A in 2006 but these were reduced in 2008 when the vaccine virus had a site A mutation. A better understanding of immunodominance may allow prediction of future antigenic drift and assist in vaccine strain selection.
| S-EPMC3405050 | biostudies-other
Project description:Seasonal epidemics caused by influenza virus are driven by antigenic changes (drift) in viral surface glycoproteins that allow evasion from preexisting humoral immunity. Antigenic drift is a feature of not only the hemagglutinin (HA), but also of neuraminidase (NA). We have evaluated the antigenic evolution of each protein in H1N1 and H3N2 viruses used in vaccine formulations during the last 15 y by analysis of HA and NA inhibition titers and antigenic cartography. As previously shown for HA, genetic changes in NA did not always lead to an antigenic change. The noncontinuous pattern of NA drift did not correspond closely with HA drift in either subtype. Although NA drift was demonstrated using ferret sera, we show that these changes also impact recognition by NA-inhibiting antibodies in human sera. Remarkably, a single point mutation in the NA of A/Brisbane/59/2007 was primarily responsible for the lack of inhibition by polyclonal antibodies specific for earlier strains. These data underscore the importance of NA inhibition testing to define antigenic drift when there are sequence changes in NA.
| S-EPMC3251064 | biostudies-literature
Project description:BackgroundHemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses. Previous studies showed that the stability of HA was significant for HA's immunogenicity, and many efforts have been made to stabilize the expressed HA proteins.MethodsIn this study, the protein disulfide isomerases (PDIs) were investigated for the ability to improve the stability of HA protein. Two members of the PDIs family, PDI and ERp57, were over-expressed or down-expressed in 293 T cells. The expression of H3 HA and PDIs were investigated by real-time qPCR, western-blot, immunofluorescence assay, and flow cytometry. The stability of HA was investigated by western-blot under non-reducing condition. Moreover, BALB/c mice were immunized subcutaneously twice with the vaccine that contained HA proteins from the ERp57-overexpressed and conventional 293 T cells respectively to investigate the impact of ERp57 on the immunogenicity of H3N2 HA.ResultsThe percentage of the disulfide-bonded HA trimers increased significantly in the PDIs-overexpressed 293 T cells, and ERp57 was more valid to the stability of HA than PDI. The knockdown of ERp57 by small interfering RNA significantly decreased the percentage of the disulfide-bonded HA trimers. HA proteins from ERp57-overexpressed 293 T cells stimulated the mice to generate significantly higher HA-specific IgG against H1N1 and H3N2 viruses than those from the conventional cells. The mice receiving H3 HA from ERp57-overexpressed 293 T cells showed the better resistance against H1N1 viruses and the higher survival rate than the mice receiving H3 HA from the conventional cells.ConclusionERp57 could improve the stability and immunogenicity of H3N2 HA.
| S-EPMC7175539 | biostudies-literature
Project description:The spread of avian H5N1 influenza viruses around the globe has become a worldwide public health concern. To evaluate the pathogenic potential of reassortant viruses between currently cocirculating avian H5N1 and human H3N2 influenza viruses, we generated all the 254 combinations of reassortant viruses between A/chicken/South Kalimantan/UT6028/06 (SK06, H5N1) and A/Tokyo/Ut-Sk-1/07 (Tok07, H3N2) influenza viruses by reverse genetics. We found that the presence of Tok07 PB2 protein in the ribonucleoprotein (RNP) complex allowed efficient viral RNA transcription in a minigenome assay and that RNP activity played an essential role in the viability and replicative ability of the reassortant viruses. When the pathogenicity of 75 reassortant H5 viruses was tested in mice, 22 were more pathogenic than the parental SK06 virus, and three were extremely virulent. Strikingly, all 22 of these viruses obtained their PB2 segment from Tok07 virus. Further analysis showed that Tok07 PB1 alone lacked the ability to enhance the pathogenicity of the reassortant viruses but could do so by cooperating with Tok07 PB2. Our data demonstrate that reassortment between an avian H5N1 virus with low pathogenicity in mice and a human virus could result in highly pathogenic viruses and that the human virus PB2 segment functions in the background of an avian H5N1 virus, enhancing its virulence. Our findings highlight the importance of surveillance programs to monitor the emergence of human H5 reassortant viruses, especially those containing a PB2 segment of human origin.
| S-EPMC2842136 | biostudies-literature
Project description:H3N2 canine influenza viruses are prevalent in Asian and North American countries. During circulation of the viruses in dogs, these viruses are occasionally transmitted to cats. If this canine virus causes an epidemic in cats too, sporadic infections may occur in humans because of the close contact between these companion animals and humans, possibly triggering an emergence of mutant viruses with a pandemic potential. In this study, we aimed to gain an insight into the mutations responsible for inter-species transmission of H3N2 virus from dogs to cats. We found that feline CRFK cell-adapted viruses acquired several mutations in multiple genome segments. Among them, HA1-K299R, HA2-T107I, NA-L35R, and M2-W41C mutations individually increased virus growth in CRFK cells. With a combination of these mutations, virus growth further increased not only in CRFK cells but also in other feline fcwf-4 cells. Both HA1-K299R and HA2-T107I mutations increased thermal resistance of the viruses. In addition, HA2-T107I increased the pH requirement for membrane fusion. These findings suggest that the mutations, especially the two HA mutations, identified in this study, might be responsible for adaptation of H3N2 canine influenza viruses in cats.
| S-EPMC6786582 | biostudies-literature
Project description:BackgroundDuring the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection.Methods and findingsTo study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject.ConclusionThe presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.
| S-EPMC3198447 | biostudies-literature