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Identification and structural characterization of a broadly neutralizing antibody targeting a novel conserved epitope on the influenza virus H5N1 hemagglutinin.

ABSTRACT: The unabated circulation of the highly pathogenic avian influenza A virus/H5N1 continues to be a serious threat to public health worldwide. Because of the high frequency of naturally occurring mutations, the emergence of H5N1 variants with high virulence has raised great concerns about the potential transmissibility of the virus in humans. Recent studies have shown that laboratory-mutated or reassortant H5N1 viruses could be efficiently transmitted among mammals, particularly ferrets, the best animal model for humans. Thus, it is critical to establish effective strategies to combat future H5N1 pandemics. In this study, we identified a broadly neutralizing monoclonal antibody (MAb), HA-7, that potently neutralized all tested strains of H5N1 covering clades 0, 1, 2.2, 2.3.4, and 2.3.2.1 and completely protected mice against lethal challenges of H5N1 viruses from clades 1 and 2.3.4. HA-7 specifically targeted the globular head of the H5N1 virus hemagglutinin (HA). Using electron microscopy technology with three-dimensional reconstruction (3D-EM), we discovered that HA-7 bound to a novel and highly conserved conformational epitope that was centered on residues 81 to 83 and 117 to 122 of HA1 (H5 numbering). We further demonstrated that HA-7 inhibited viral entry during postattachment events but not at the receptor-binding step, which is fully consistent with the 3D-EM result. Taken together, we propose that HA-7 could be humanized as an effective passive immunotherapeutic agent for antiviral stockpiling for future influenza pandemics caused by emerging unpredictable H5N1 strains. Our study also provides a sound foundation for the rational design of vaccines capable of inducing broad-spectrum immunity against H5N1.

SUBMITTER: Du L

PROVIDER: S-EPMC3571485 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Identification and structural characterization of a broadly neutralizing antibody targeting a novel conserved epitope on the influenza virus H5N1 hemagglutinin.

Du Lanying L Jin Lei L Zhao Guangyu G Sun Shihui S Li Junfeng J Yu Hong H Li Ye Y Zheng Bo-Jian BJ Liddington Robert C RC Zhou Yusen Y Jiang Shibo S

Journal of virology 20121205 4

The unabated circulation of the highly pathogenic avian influenza A virus/H5N1 continues to be a serious threat to public health worldwide. Because of the high frequency of naturally occurring mutations, the emergence of H5N1 variants with high virulence has raised great concerns about the potential transmissibility of the virus in humans. Recent studies have shown that laboratory-mutated or reassortant H5N1 viruses could be efficiently transmitted among mammals, particularly ferrets, the best a ...[more]

PMID: 23221567

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Project description:UnlabelledNeutralizing 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.ImportanceAntibodies 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.

Project description:Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

Dataset Information

Identification and structural characterization of a broadly neutralizing antibody targeting a novel conserved epitope on the influenza virus H5N1 hemagglutinin.

Publications

Identification and structural characterization of a broadly neutralizing antibody targeting a novel conserved epitope on the influenza virus H5N1 hemagglutinin.

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