A Human Antibody Recognizing a Conserved Epitope of H5 Hemagglutinin Broadly Neutralizes Highly Pathogenic Avian Influenza H5N1 Viruses

Abstract

e Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotypebased neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains.

Divergent Requirement of Fc-Fcγ Receptor Interactions for In Vivo Protection against Influenza Viruses by Two Pan-H5 Hemagglutinin Antibodies

“…These Abs potently neutralize all clades and subclades of HPAI H5N1 viruses except subclade 7.2. Furthermore, they protect against lethal challenge of both homologous and heterologous H5N1 strains (26,27). More recently, we showed that 65C6 and 100F4 Abs also cross-neutralize newly emerging HPAI H5N6 and H5N8 reassortants.…”

“…In addition, Abs against the globular head with different degrees of cross-reactivity have also been isolated (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Many of these Abs are virus strain specific and recognize epitopes located in the receptor binding site (RBS), but some Abs recognize conserved epitopes within or outside the RBS of diverse strains of different subtypes (17)(18)(19) or within a HA subtype (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The antibody repertoire against epitopes located in the head is more diverse than those Abs targeting epitopes in the stem (31).…”

The Vestigial Esterase Domain of Haemagglutinin of H5N1 Avian Influenza A Virus: Antigenicity and Contribution to Viral Pathogenesis

“…Significant abrogation of binding in western blot analysis was observed when WLLGNP was mutated to WRRGNP. Another human mAb, 100F4, neutralises multiple clades of H5N1 and binds outside the receptor binding subdomain of H5N1 HA [12,22]. Memory B cells from peripheral blood mononuclear cells of a patient who recovered from H5N1 infection were immortalised and culture supernatants were screened by HA pseudotype-based neutralisation assay.…”

Phage and Yeast Display

“…Broad domain mapping for independent panels of anti-influenza mAbs are described from yeast libraries displaying fulllength, precursor influenza HA (HA0) or the HA1 and HA2 subunits separately (113,114). After confirming binding and domain localization, sublibraries of mutant HA fragments were generated by error-prone PCR for fine epitope mapping.…”