Yoshitaka Iba

Shiga toxin 2 (Stx2)-specific mAb-producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)-specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat-labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)-specific whereas five were Stx2B-specific antibody-producing clones. The in vitro neutralization activity of Stx2B-specific mAbs against Stx2 was greater than that of Stx2A-specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B-specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B-specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the V-H and V-L regions of clones 45 and 75D9 were determined. Our Stx2B-specific mAbs may be new candidates for the development of mouse-human chimeric Stx2-neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.

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.

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 V-H 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.