Defining influenza a virus hemagglutinin antigenic drift by sequential monoclonal antibody selection.
ABSTRACT Human influenza A virus (IAV) vaccination is limited by "antigenic drift," rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined by monoclonal or polyclonal Abs. Sequential mutants grow robustly, showing the structural plasticity of HA, although several hemagglutinin substitutions required an epistatic substitution in the neuraminidase glycoprotein to maximize growth. Selecting escape mutants from parental versus sequential variants with the same mAb revealed distinct escape repertoires, attributed to contextual changes in antigenicity and the mutation landscape. Since each hemagglutinin mutation potentially sculpts future mutation space, drift can follow many stochastic paths, undermining its unpredictability and underscoring the need for drift-insensitive vaccines.
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ABSTRACT: Human gammaherpesviruses are associated with the development of lymphomas and epithelial malignancies. The heterogeneity of these tumors reflects the ability of these viruses to route infection to different cell types at various stages of their lifecycle. While the Epstein Barr virus uses gp42 - human leukocyte antigen class II interaction as a switch of cell tropism, the molecular mechanism that orientates tropism of rhadinoviruses is still poorly defined. Here, we used bovine herpesvirus 4 (BoHV-4) to further elucidate how rhadinoviruses regulate their infectivity. In the absence of any gp42 homolog, BoHV-4 exploits the alternative splicing of its Bo10 gene to produce distinct viral populations that behave differently based on the originating cell. While epithelial cells produce virions with high levels of the accessory envelope protein gp180, encoded by a Bo10 spliced product, myeloid cells express reduced levels of gp180. As a consequence, virions grown in epithelial cells are hardly infectious for CD14+ circulating cells, but are relatively resistant to antibody neutralization due to the shielding property of gp180 for vulnerable entry epitopes. In contrast, myeloid virions readily infect CD14+ circulating cells but are easily neutralized. This molecular switch could therefore allow BoHV-4 to promote either, on the one hand, its dissemination into the organism, or, on the other hand, its transmission between hosts.PLoS Pathogens 10/2013; 9(10):e1003753. DOI:10.1371/journal.ppat.1003753 · 8.06 Impact Factor
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ABSTRACT: The 2009 H1N1 lineage represented the first detection of a novel, highly transmissible influenza A virus genotype: six gene segments originated from the North American triple-reassortant swine lineage; two segments, NA and M, derived from the Eurasian avian-like swine lineage. As neither parental lineage transmits efficiently between humans, the adaptations and mechanisms underlying the pandemic spread of the swine-origin 2009 strain are not clear. Toward identifying determinants of transmission, we used reverse genetics to introduce gene segments of an early pandemic isolate, A/Netherlands/602/2009 [H1N1] (NL602), into the background of A/Puerto Rico/8/1934 [H1N1] (PR8) and evaluated the resultant viruses in a guinea pig transmission model. Whereas the NL602 virus spread efficiently, the PR8 virus did not transmit. Swapping of the HA, NA and M segments of NL602 into the PR8 background yielded a virus with indistinguishable contact transmissibility to the wild-type pandemic strain. Consistent with earlier reports, the pandemic M segment alone accounted for much of the improvement in transmission. Toward understanding how the M segment might affect transmission, we evaluated neuraminidase activity and virion morphology of reassortant viruses. Transmission was found to correlate with higher neuraminidase activity and a more filamentous morphology. Importantly, we found that introduction of the pandemic M segment alone resulted in an increase in the neuraminidase activity of two pairs of otherwise isogenic PR8-based viruses. Thus, our data demonstrate the surprising result that functions encoded by the influenza A virus M segment impact neuraminidase activity and, perhaps through this mechanism, have a potent effect on transmissibility.Importance Our work uncovers a previously unappreciated mechanism through which the influenza A virus M segment can alter the receptor destroying activity of an influenza virus. Concomitant with changes to neuraminidase activity, the M segment impacts the morphology of the influenza A virion and transmissibility of the virus in the guinea pig model. We suggest that changes in NA activity underlie the ability of the influenza M segment to influence virus transmissibility. Furthermore, we show that co-adapted M, NA, and HA segments are required to provide optimal transmissibility to an influenza virus. The M-NA functional interaction we describe appears to underlie the prominent role of the 2009 pandemic M segment in supporting efficient transmission, and may be a highly important means by which influenza A viruses restore HA/NA balance following reassortment or transfer to new host environments.Journal of Virology 01/2014; DOI:10.1128/JVI.03607-13 · 4.65 Impact Factor
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ABSTRACT: There is an urgent need for a rapid diagnostic system to detect the H5 subtype of the influenza A virus. We previously developed monoclonal antibodies (mAbs) against the H5 hemagglutinin (HA) for use in a rapid diagnostic kit. In this study, we determined the epitopes of the anti-H5 HA murine mAbs OM-b, AY-2C2, and YH-1A1. Binding assays of the mAbs to different strains of H5 HAs indicated that OM-b and AY-2C2 cross-reacted with HAs from clades 1, 18.104.22.168, 2.2, and 2.3.4, whereas YH-1A1 failed to bind to those of clades 22.214.171.124 and 2.3.4. HA chimeras revealed that the epitopes for each of the mAbs were in the HA1 region. Analysis of escape mutants revealed that OM-b and AY-2C2 mAbs interacted mainly with amino acid residues D43 and G46, and the YH-1A1 mAb interacted with G139 and K or R140 of H5 HA. Multiple alignments of H5 HA protein sequences showed that D43 and G46 were very conserved among H5N1 HAs, except those in clade 2.2.1 and clade 7 (88.7%). The epitope for YH-1A1 mAb was highly variable in the HAs of H5N1, although it was well conserved in those of H5N2-N9. The OM-b and AY-2C2 mAbs could bind to the HAs of clades 1.1 and 126.96.36.199 that are currently epidemic in Asia, and we conclude that these would be effective for the detection of H5N1 infections in this region.PLoS ONE 06/2014; 9(6):e99201. DOI:10.1371/journal.pone.0099201 · 3.53 Impact Factor