Unraveling of a Neutralization Mechanism by Two Human Antibodies against Conserved Epitopes in the Globular Head of H5 Hemagglutinin

Abstract

The rapid spread of highly pathogenic avian influenza (HPAI) H5N1 virus underscores the importance of effective antiviral treatment. Previously, we developed human monoclonal antibodies 65C6 and 100F4 that neutralize almost all (sub)clades of HPAI H5N1. The conserved 65C6 epitope was mapped to the globular head of HA. However, neither the 100F4 epitope nor the neutralization mechanism by these antibodies was known. In this study, we determined the 100F4 epitope and unraveled a neutralization mechanism by antibodies 65C6 and 100F4. Influenza A virus infection is a persistent threat to public health worldwide. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. Previously, we developed human monoclonal antibodies (MAb) 65C6 and 100F4 that potently neutralize all H5 clades and subclades of highly pathogenic avian influenza (HPAI) H5N1 viruses except subclade 7.2 (Fig. 1A) and defined a conformational 65C6 epitope (1). In this study, we determined the 100F4 epitope and dissected the neutralization mechanism by these antibodies.To map the 100F4 epitope, a yeast display analysis was carried out similarly to the way we mapped the 65C6 epitope (1, 2). Figure 1B shows the 15 single amino acid mutations in H5 hemagglutinin (HA) that abolish the binding of antibody 100F4. Among these, the 7 residues at positions 68,112, 137, 143, 251, 254, and 255 were on the HA surface, while the rest were underneath the surface.To test whether these 7 surface mutations would affect neutralization by antibody 100F4, genes encoding 7 full-length H5 HA single mutants derived from H5N1 strain A/Beijing/01/03 subclade 7.1 were constructed and used to generate H5N1 pseudotypes. The resistance of H5N1 pseudotypes to antibody 100F4 was measured with the pseudotype-based neutralization assay (3). Compared to the wild-type subclade 7.1 H5N1 pseudotype, only H5N1 pseudotypes expressing H5 HA mutants with mutations at position 68 or 112 (72 or 116 according to H3 numbering) were dramatically resistant to antibody 100F4 (Fig. 1C and D). On the HA surface, these two resistant residues are adjacent to each other (Fig. 1E), but they are next to the Cb in H1 HA and site E in H3 HA (4-7) ( Fig. 1F and G). The 100F4 epitope does not overlap any known epitopes in the head region detected by human and mouse MAb ( Fig. 1H and I). Thus, the 100F4 epitope is a new conserved conformational epitope on the globular head and away from the receptor binding site (RBS). In contrast, the 65C6 epitope partially overlaps with Sa in H1 HA at residue 161 (K165 according to H3 numbering) and with site A in H3 HA at residues 118 and 121 (T122 and F125 according to H3 numbering) (4-7). In addition, the 65C6 epitope also partially overlaps epitopes detected by some human MAb, i.e., FLA5.10 at P118 (P122 according to H3 numbering), FLD21.140 at S121, Y164, and T167 (S125, Y168, and T171 according to H3 numbering) (8), AVFLuigG01 at P118, Y164, and T167 (P122, ...

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

“…When tested in vitro, 100F4 was able to directly neutralise A/Shenzhen/406H/2006 and A/chicken/Netherlands/14015526/2014 [29] by binding to IAV and preventing low-pH triggered membrane fusion after entry into the cell with IAV [12]. 46B8 was also able to neutralise IBV via the same mechanism.…”

“…Culture supernatants were harvested, and the human monoclonal antibodies in these culture supernatants were purified. 100F4 was reported to bind to amino acid residues at positions D77 and E119 which are found in the VE subdomain of H5N1 HA [12] (Fig. 5).…”

“…Interestingly, several mAbs in this class have been documented to bind to the vestigial esterase (VE) subdomain of HA of the H5N1 subtype and they have been shown to bind to multiple clades of H5N1 viruses [11][12][13][14][15]. However, they typically do not bind to other subtypes of IAV which suggests that the VE subdomain is conserved within a subtype but diverse among subtypes.…”

“…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.…”

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

“…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).…”