Nobuko Ohshima

Naturally Occurring Antibodies in Humans Can Neutralize a Variety of Influenza Virus Strains, Including H3, H1, H2, and H5

ABSTRACT Influenza A viruses are classified into 16 subtypes according to the serotypes of hemagglutinin (HA). It is generally thought that neutralizing antibodies (Abs) are not broadly cross-reactive among HA subtypes. We examined the repertoire of neutralizing Abs against influenza viruses in humans. B lymphocytes were collected from donors by apheresis, and Ab libraries were constructed by using phage-display technology. Anti-HA clones were isolated by screening with H3N2 viruses. Their binding activity was examined, and four kinds of Abs showing broad strain specificity were identified from one donor. Two of the Abs, F045-092 and F026-427, were extensively analyzed. They neutralized not only H3N2 but also H1N1, H2N2, and H5N1 viruses, although the activities were largely varied. Flow cytometry suggested that they have the ability to bind to HA and HA1 artificially expressed on the cell surface. They show hemagglutination inhibition activity and do not compete with C179, an Ab thought to bind to the stalk region. F045-092 competes with Abs that recognize sites A and B for binding to HA. Furthermore, the serine at residue 136 in site A could be a part of the epitope. Thus, it is likely that F045-092 and F026-427 bind to a conserved epitope in the head region formed by HA1. Interestingly, while the VH1-69 gene can encode MAbs against the HA stem that are group 1 specific, F045-092 and its relatives that recognize the head region also use VH1-69. The possible epitope recognized by these clones is discussed.

Receptor mimicry by antibody F045–092 facilitates universal binding to the H3 subtype of influenza virus

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.

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

ABSTRACT 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. IMPORTANCE Antibodies 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.

Monoclonal antibodies in man that neutralized H3N2 influenza viruses were classified into three groups with distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003.

We tried to reveal the strain specificity of neutralizing mAbs against H3N2 influenza viruses in individuals. A large number of B lymphocytes of a pediatrician were collected by apheresis and two Ab libraries were constructed at 2004 and 2007 by using the phage-display technology. The libraries were screened against 12 different H3 strains of flu isolated between 1968 and 2004. Large numbers of clones that bound to the Ags were isolated and mAbs that specifically bound to H3 strain viruses were selected. Their binding activity to the 12 strains and neutralizing activity were studied by ELISA and focus reduction test, respectively. Furthermore, the binding activity to hemagglutinin (HA) was examined by Western blot. The majority of clones showing the neutralizing activity turned out to be anti-HA mAbs and could be divided into three major groups showing distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003.

Localization of epitopes recognized by monoclonal antibodies that neutralized the H3N2 influenza viruses in man.

Through extensive isolation of neutralizing mAbs against H3N2 influenza viruses representing the in vivo repertoire in a human donor, we examined the relationships between antigenic drift of influenza virus and protective antibodies generated in an infected individual. The majority of mAbs isolated from a donor born in 1960 were divided into three major groups with distinct strain specificity: 1968–1973, 1977–1993 and 1997–2003. In the present study, we developed a new method that allowed us to comprehensively determine the location of epitopes recognized by many mAbs. Original haemagglutinins (HAs) of several strains and chimaeric variants, in which one of the seven sites (A, B1, B2, C1, C2, D or E) was replaced by some other strain-derived sequence, were artificially expressed on the cell surface. The binding activity of mAbs to the HAs was examined by flow cytometry. By using this method, we determined the location of epitopes recognized by 98 different mAbs. Clones that neutralize the 1968–1973 strains bind to site B2/D, A or A/B1. While sites C, E and B were recognized by clones that neutralized the 1977–1993 strains, the majority of these clones bind to site C. Clones that neutralize the 1997–2003 strains bind to site B, A/B1, A/B2 or E/C2.

Two Types of Antibodies Are Induced by Vaccination with A/California/2009pdm Virus: Binding near the Sialic Acid-Binding Pocket and Neutralizing Both H1N1 and H5N1 Viruses

Many people have a history of catching the flu several times during childhood but no additional flu in adulthood, even without vaccination. We analyzed the total repertoire of antibodies (Abs) against influenza A group 1 viruses induced in such a flu-resistant person after vaccination with 2009 H1N1 pandemic influenza virus. They were classified into two types, with no exceptions. The first type, the products of B cells newly induced through vaccination, binds near the sialic acid-binding pocket. The second type, the products of long-lived memory B cells established before vaccination, utilizes the 1-69 VH gene, binds to the stem of HA, and neutralizes both H1N1 and H5N1 viruses with few exceptions. These observations indicate that the sialic acid-binding pocket and its surrounding region are immunogenically very potent and majority of the B cells whose growth is newly induced by vaccination produce Abs that recognize these regions. However, they play a role in protection against influenza virus infection for a short period since variant viruses that have acquired resistance to these Abs become dominant. On the other hand, although the stem of HA is immunogenically not potent, the second type of B cells eventually becomes dominant. Thus, a selection system should function in forming the repertoire of long-lived memory B cells and the stability of the epitope would greatly affect the fate of the memory cells. Acquisition of the ability to produce Abs that bind to the stable epitope could be a major factor of flu resistance.

Three Types of Broadly Reacting Antibodies against Influenza B Viruses Induced by Vaccination with Seasonal Influenza Viruses

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.