Nobuko Ohshima

Four kinds of avian-derived H5N1 influenza virus, A/Vietnam/1194/2004 (Clade 1), A/Indonesia/5/2005 (Clade 2.1), A/Qinghai/1A/2005 (Clade 2.2), and A/Anhui/1/2005 (Clade 2.3), have been stocked in Japan for use as pre-pandemic vaccines. When a pandemic occurs, these viruses would be used as vaccines in the hope of inducing immunity against the pandemic virus. We analyzed the specificity of antibodies (Abs) produced by B lymphocytes present in the blood after immunization with these vaccines. Eighteen volunteers took part in this project. After libraries of Ab-encoding sequences were constructed using blood from subjects vaccinated with these viruses, a large number of clones that encoded Abs that bound to the virus particles used as vaccines were isolated. These clones were classified into two groups according to the hemagglutination inhibition (HI) activity of the encoded Abs. While two-thirds of the clones were HI positive, the encoded Abs exhibited only restricted strain specificity. On the other hand, half of the HI-negative clones encoded Abs that bound not only to the H5N1 virus but also to the H1N1 virus; with a few exceptions, these Abs appeared to be encoded by memory B cells present before vaccination. The HI-negative clones included those encoding broadly cross-reactive Abs, some of which were encoded by non-VH1-69 germline genes. However, although this work shows that various kinds of anti-H5N1 Abs are encoded by volunteers vaccinated with pre-pandemic vaccines, broad cross-reactivity was seen only in a minority of clones, raising concern regarding the utility of these H5N1 vaccine viruses for the prevention of H5N1 pandemics.

We analyzed the antibody (Ab) repertoire against influenza B viruses induced by vaccination with seasonal influenza viruses in one individual who had never been vaccinated until 2009. The vaccine used in this study comprised B/Massachusetts/2/2012 (Yamagata lineage), A/Texas/50/2012 (H3N2), and A/California/7/2009 (H1N1). One month after the subject received two vaccinations, blood (200 ml) was obtained and peripheral mononuclear cells were prepared, and a large Ab library was constructed using phage display technology. The library was screened with HA-enriched fraction of B/Massachusetts/2/2012 and B/Brisbane/60/2008 (Victoria lineage) virus, and a total of 26 Abs that potentially bound to hemagglutinin (HA) molecules were isolated. Their binding activities to six influenza B viruses, three of Yamagata lineage and three of Victoria lineage, and two influenza A viruses, H1N1 and H3N2, were examined. The Abs showed cross-reactivity at three different levels. The first type bound to all Yamagata lineage viruses. The second type bound to both Yamagata and Victoria lineage viruses. The third type bound to both influenza A and B viruses. These results indicate that common epitopes exist on HA molecules of influenza virus at various levels, and humans have capability to produce Abs that bind to such common epitopes.

Neutralizing 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.

Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012-2013. Here we describe an antibody, F045-092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045-092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045-092 extends its recognition to divergent subtypes, including H1, H2 and H13, using the enhanced avidity of its IgG to overcome lower-affinity Fab binding, as observed with other antibodies that target the receptor-binding site. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small-molecule therapeutics.