Immunogenic Stimulus for Germline Precursors of Antibodies that Engage the Influenza Hemagglutinin Receptor-Binding Site.
ABSTRACT: Influenza-virus antigenicity evolves to escape host immune protection. Antibody lineages within individuals evolve in turn to increase affinity and hence potency. Strategies for a "universal" influenza vaccine to elicit lineages that escape this evolutionary arms race and protect against seasonal variation and novel, pandemic viruses will require directing B cell ontogeny to focus the humoral response on conserved epitopes on the viral hemagglutinin (HA). The unmutated common ancestors (UCAs) of six distinct, broadly neutralizing antibody lineages from one individual bind the HA of a virus circulating at the time the participant was born. HAs of viruses circulating more than 5 years later no longer bind the UCAs, but mature antibodies in the lineages bind strains from the entire 18-year lifetime of the participant. The analysis shows how immunological memory shaped the response to subsequent influenza exposures and suggests that early imprinting by a suitable influenza antigen may enhance likelihood of later breadth.
Project description:Influenza viruses use distinct antibody escape mechanisms depending on the overall complexity of the antibody response that is encountered. When grown in the presence of a hemagglutinin (HA) monoclonal antibody, influenza viruses typically acquire a single HA mutation that reduces the binding of that specific monoclonal antibody. In contrast, when confronted with mixtures of HA monoclonal antibodies or polyclonal sera that have antibodies that bind several HA epitopes, influenza viruses acquire mutations that increase HA binding to host cells. Recent data from our laboratory and others suggest that some humans possess antibodies that are narrowly focused on HA epitopes that were present in influenza virus strains that they were likely exposed to in childhood. Here, we completed a series of experiments to determine if humans with narrowly focused HA antibody responses are able to select for influenza virus antigenic escape variants in ovo We identified three human donors that possessed HA antibody responses that were heavily focused on a single HA antigenic site. Sera from all three of these donors selected single HA escape mutations during in ovo passage experiments, similar to what has been previously reported for single monoclonal antibodies. These single HA mutations directly reduced binding of serum antibodies used for selection. We propose that new antigenic variants of influenza viruses might originate in individuals who produce antibodies that are narrowly focused on HA epitopes that were present in viral strains that they encountered in childhood.IMPORTANCE Influenza vaccine strains must be updated frequently since circulating viral strains continuously change in antigenically important epitopes. Our previous studies have demonstrated that some individuals possess antibody responses that are narrowly focused on epitopes that were present in viral strains that they encountered during childhood. Here, we show that influenza viruses rapidly escape this type of polyclonal antibody response when grown in ovo by acquiring single mutations that directly prevent antibody binding. These studies improve our understanding of how influenza viruses evolve when confronted with narrowly focused polyclonal human antibodies.
Project description:Circulating influenza viruses evade neutralization in their human hosts by acquiring escape mutations at epitopes of prevalent antibodies. A goal for next-generation influenza vaccines is to reduce escape likelihood by selectively eliciting antibodies recognizing conserved surfaces on the viral hemagglutinin (HA). The receptor-binding site (RBS) on the HA "head" and a region near the fusion peptide on the HA "stem" are two such sites. We describe here a human antibody clonal lineage, designated CL6649, members of which bind a third conserved site ("lateral patch") on the side of the H1-subtype, HA head. A crystal structure of HA with bound Fab6649 shows the conserved antibody footprint. The site was invariant in isolates from 1977 (seasonal) to 2012 (pdm2009); antibodies in CL6649 recognize HAs from the entire period. In 2013, human H1 viruses acquired mutations in this epitope that were retained in subsequent seasons, prompting modification of the H1 vaccine component in 2017. The mutations inhibit Fab6649 binding. We infer from the rapid spread of these mutations in circulating H1 influenza viruses that the previously subdominant, conserved lateral patch had become immunodominant for individuals with B-cell memory imprinted by earlier H1 exposure. We suggest that introduction of the pdm2009 H1 virus, to which most of the broadly prevalent, neutralizing antibodies did not bind, conferred a selective advantage in the immune systems of infected hosts to recall of memory B cells that recognized the lateral patch, the principal exposed epitope that did not change when pdm2009 displaced previous seasonal H1 viruses.
Project description:The emergence and seasonal persistence of pathogenic H7N9 influenza viruses in China have raised concerns about the pandemic potential of this strain, which, if realized, would have a substantial effect on global health and economies. H7N9 viruses are able to bind to human sialic acid receptors and are also able to develop resistance to neuraminidase inhibitors without a loss in fitness. It is not clear whether prior exposure to circulating human influenza viruses or influenza vaccination confers immunity to H7N9 strains. Here, we demonstrate that 3 of 83 H3 HA-reactive monoclonal antibodies generated by individuals that had previously undergone influenza A virus vaccination were able to neutralize H7N9 viruses and protect mice against homologous challenge. The H7N9-neutralizing antibodies bound to the HA stalk domain but exhibited a difference in their breadth of reactivity to different H7 influenza subtypes. Mapping viral escape mutations suggested that these antibodies bind at least two different epitopes on the stalk region. Together, these results indicate that these broadly neutralizing antibodies may contribute to the development of therapies against H7N9 strains and may also be effective against pathogenic H7 strains that emerge in the future.
Project description:Influenza B viruses make a considerable contribution to morbidity attributed to seasonal influenza. Currently circulating influenza B isolates are known to belong to two antigenically distinct lineages referred to as B/Victoria and B/Yamagata. Frequent exchange of genomic segments of these two lineages has been noted in the past, but the observed patterns of reassortment have not been formalized in detail. We investigate interlineage reassortments by comparing phylogenetic trees across genomic segments. Our analyses indicate that of the eight segments of influenza B viruses only segments coding for polymerase basic 1 and 2 (PB1 and PB2) and hemagglutinin (HA) proteins have maintained separate Victoria and Yamagata lineages and that currently circulating strains possess PB1, PB2, and HA segments derived entirely from one or the other lineage; other segments have repeatedly reassorted between lineages thereby reducing genetic diversity. We argue that this difference between segments is due to selection against reassortant viruses with mixed-lineage PB1, PB2, and HA segments. Given sufficient time and continued recruitment to the reassortment-isolated PB1-PB2-HA gene complex, we expect influenza B viruses to eventually undergo sympatric speciation.
Project description:Protein thermodynamics are an integral determinant of viral fitness and one of the major drivers of protein evolution. Mutations in the influenza A virus (IAV) hemagglutinin (HA) protein can eliminate neutralizing antibody binding to mediate escape from preexisting antiviral immunity. Prior research on the IAV nucleoprotein suggests that protein stability may constrain seasonal IAV evolution; however, the role of stability in shaping the evolutionary dynamics of the HA protein has not been explored. We used the full coding sequence of 9,797 H1N1pdm09 HA sequences and 16,716 human seasonal H3N2 HA sequences to computationally estimate relative changes in the thermal stability of the HA protein between 2009 and 2016. Phylogenetic methods were used to characterize how stability differences impacted the evolutionary dynamics of the virus. We found that pandemic H1N1 IAV strains split into two lineages that had different relative HA protein stabilities and that later variants were descended from the higher-stability lineage. Analysis of the mutations associated with the selective sweep of the higher-stability lineage found that they were characterized by the early appearance of highly stabilizing mutations, the earliest of which was not located in a known antigenic site. Experimental evidence further suggested that H1N1 HA stability may be correlated with in vitro virus production and infection. A similar analysis of H3N2 strains found that surviving lineages were also largely descended from viruses predicted to encode more-stable HA proteins. Our results suggest that HA protein stability likely plays a significant role in the persistence of different IAV lineages. IMPORTANCE One of the constraints on fast-evolving viruses, such as influenza virus, is protein stability, or how strongly the folded protein holds together. Despite the importance of this protein property, there has been limited investigation of the impact of the stability of the influenza virus hemagglutinin protein-the primary antibody target of the immune system-on its evolution. Using a combination of computational estimates of stability and experiments, our analysis found that viruses with more-stable hemagglutinin proteins were associated with long-term persistence in the population. There are two potential reasons for the observed persistence. One is that more-stable proteins tolerate destabilizing mutations that less-stable proteins could not, thus increasing opportunities for immune escape. The second is that greater stability increases the fitness of the virus through increased production of infectious particles. Further research on the relative importance of these mechanisms could help inform the annual influenza vaccine composition decision process.
Project description:To determine if the influenza B virus HA is under constraints that limit its antigenic variation, we performed a transposon screen to compare the mutational tolerance of the currently circulating influenza A virus HAs (H1 and H3 subtypes) and influenza B virus HAs (B/Victoria87 and B/Yamagata88 antigenic lineages). A library of insertional mutants for each HA was generated and deep sequenced after passaging to determine where insertions were tolerated in replicating viruses. Overall design: RNA-seq of influenza virus HA libraries
Project description:In nearly all characterized influenza viruses, hemagglutinin (HA) is the receptor-binding protein while neuraminidase (NA) is a receptor-cleaving protein that aids in viral release. However, in recent years, several groups have described point mutations that confer receptor-binding activity on NA, albeit in laboratory rather than natural settings. One of these mutations, D151G, appears to arise in the NA of recent human H3N2 viruses upon passage in tissue culture. We inadvertently isolated the second of these mutations, G147R, in the NA of the lab-adapted A/WSN/33 (H1N1) strain while we were passaging a heavily engineered virus in the lab. G147R also occurs at low frequencies in the reported sequences of viruses from three different lineages: human 2009 pandemic H1N1 (pdmH1N1), human seasonal H1N1, and chicken H5N1. Here we reconstructed a representative G147R NA from each of these lineages and found that all of the proteins have acquired the ability to bind an unknown cellular receptor while retaining substantial sialidase activity. We then reconstructed a virus with the HA and NA of a reported G147R pdmH1N1 variant and found no attenuation of viral replication in cell culture or change in pathogenesis in mice. Furthermore, the G147R virus had modestly enhanced resistance to neutralization by the Fab of an antibody against the receptor-binding pocket of HA, although it remained completely sensitive to the full-length IgG. Overall, our results suggest that circulating N1 viruses occasionally may acquire the G147R NA receptor-binding mutation without impairment of replicative capacity.Influenza viruses have two main proteins on their surface: one (hemagglutinin) binds incoming viruses to cells, while the other (neuraminidase) helps release newly formed viruses from these same cells. Here we characterize unusual mutant neuraminidases that have acquired the ability to bind to cells. We show that the mutation that allows neuraminidase to bind cells has no apparent adverse effect on viral replication but does make the virus modestly more resistant to a fragment of an antibody that blocks the normal hemagglutinin-mediated mode of viral attachment. Our results suggest that viruses with receptor-binding neuraminidases may occur at low levels in circulating influenza virus lineages.
Project description:Phylogenetic profiles of the genes coding for the hemagglutinin (HA) protein, nucleoprotein (NP), matrix (M) protein, and nonstructural (NS) proteins of influenza B viruses isolated from 1940 to 1998 were analyzed in a parallel manner in order to understand the evolutionary mechanisms of these viruses. Unlike human influenza A (H3N2) viruses, the evolutionary pathways of all four genes of recent influenza B viruses revealed similar patterns of genetic divergence into two major lineages. Although evolutionary rates of the HA, NP, M, and NS genes of influenza B viruses were estimated to be generally lower than those of human influenza A viruses, genes of influenza B viruses demonstrated complex phylogenetic patterns, indicating alternative mechanisms for generation of virus variability. Topologies of the evolutionary trees of each gene were determined to be quite distinct from one another, showing that these genes were evolving in an independent manner. Furthermore, variable topologies were apparently the result of frequent genetic exchange among cocirculating epidemic viruses. Evolutionary analysis done in the present study provided further evidence for cocirculation of multiple lineages as well as sequestering and reemergence of phylogenetic lineages of the internal genes. In addition, comparison of deduced amino acid sequences revealed a novel amino acid deletion in the HA1 domain of the HA protein of recent isolates from 1998 belonging to the B/Yamagata/16/88-like lineage. It thus became apparent that, despite lower evolutionary rates, influenza B viruses were able to generate genetic diversity among circulating viruses through a combination of evolutionary mechanisms involving cocirculating lineages and genetic reassortment by which new variants with distinct gene constellations emerged.
Project description:Here, we address the question of why the influenza A virus hemagglutinin (HA) does not escape immunity by hyperglycosylation. Uniquely among dozens of monoclonal antibodies specific for A/Puerto Rico/8/34, escape from H28-A2 neutralization requires substitutions introducing N-linked glycosylation at residue 131 or 144 in the globular domain. This escape decreases viral binding to cellular receptors, which must be compensated for by additional substitutions in HA or neuraminidase that enable viral replication. Sequence analysis of circulating H1 influenza viruses confirms the in vivo relevance of our findings: natural occurrence of glycosylation at residue 131 is always accompanied by a compensatory mutation known to increase HA receptor avidity. In vaccinated mice challenged with WT vs. H28-A2 escape mutants, the selective advantage conferred by glycan-mediated global reduction in antigenicity is trumped by the costs of diminished receptor avidity. These findings show that, although N-linked glycosylation can broadly diminish HA antigenicity, fitness costs restrict its deployment in immune evasion.
Project description:Mutations arise in the genomes of progeny viruses during infection. Mutations that occur in epitopes targeted by host antibodies allow the progeny virus to escape the host adaptive, B-cell mediated antibody immune response. Major epitopes have been identified in influenza B virus (IBV) hemagglutinin (HA) protein. However, IBV strains maintain a seasonal presence in the human population and changes in IBV genomes in response to immune pressure are not well characterized. There are two lineages of IBV that have circulated in the human population since the 1980s, B-Victoria and B-Yamagata. It is hypothesized that early exposure to one influenza subtype leads to immunodominance. Subsequent seasonal vaccination or exposure to new subtypes may modify subsequent immune responses, which, in turn, results in selection of escape mutations in the viral genome. Here we show that while some mutations do occur in known epitopes suggesting antibody escape, many mutations occur in other parts of the HA protein. Analysis of mutations outside of the known epitopes revealed that these mutations occurred at the same amino acid position in viruses from each of the two IBV lineages. Interestingly, where the amino acid sequence differed between viruses from each lineage, reciprocal amino acid changes were observed. That is, the virus from the Yamagata lineage become more like the Victoria lineage virus and vice versa. Our results suggest that some IBV HA sequences are constrained to specific amino acid codons when viruses are cultured in the presence of antibodies. Some changes to the known antigenic regions may also be restricted in a lineage-dependent manner. Questions remain regarding the mechanisms underlying these results. The presence of amino acid residues that are constrained within the HA may provide a new target for universal vaccines for IBV.