The Epitope and Neutralization Mechanism of AVFluIgG01, a Broad-Reactive Human Monoclonal Antibody against H5N1 Influenza Virus

Abstract

The continued spread of highly pathogenic avian influenza (HPAI) H5N1 virus underscores the importance of effective antiviral approaches. AVFluIgG01 is a potent and broad-reactive H5N1-neutralizing human monoclonal antibody (mAb) showing great potential for use either for therapeutic purposes or as a basis of vaccine development, but its antigenic epitope and neutralization mechanism have not been finely characterized. In this study, we first demonstrated that AVFluIgG01 targets a novel conformation-dependent epitope in the globular head region of H5N1 hemagglutinin (HA). By selecting mimotopes from a random peptide library in combination with computational algorithms and site-directed mutagenesis, the epitope was mapped to three conserved discontinuous sites (I-III) that are located closely at the three-dimensional structure of HA. Further, we found that this HA1-specific human mAb can efficiently block both virus-receptor binding and post-attachment steps, while its Fab fragment exerts the post-attachment inhibition only. Consistently, AVFluIgG01 could inhibit HA-mediated cell-cell membrane fusion at a dose-dependent manner and block the acquisition of pH-induced protease sensitivity. These results suggest a neutralization mechanism of AVFluIgG01 by simultaneously blocking viral attachment to the receptors on host cells and interfering with HA conformational rearrangements associated with membrane fusion. The presented data provide critical information for developing novel antiviral therapeutics and vaccines against HPAI H5N1 virus.

A Unique and Conserved Neutralization Epitope in H5N1 Influenza Viruses Identified by an Antibody against the A/Goose/Guangdong/1/96 Hemagglutinin

“…1C). These key H5 residues do not overlap any known epitopes described above or detected with other human and mouse antibodies (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51). The four H5M9 epitope key residues do not overlap known H1 antigenic sites (36), but positions 53, 274, and 276 correspond to site C of H3 viruses (35).…”

“…In addition to preventing virus binding, H5M9 appeared to inhibit the postattachment membrane fusion step in our trypsin susceptibility test at low pH 4.9. Another anti-H5N1 antibody, AVFluIgG01, which binds to the HA1 receptor binding subdomain with potential epitope residues 123 to 125, 128, and 168, was also reported to have a similar dual neutralization mechanism (38). Interestingly, antibody CR8071, which neutralizes influenza B viruses, also bound to the vestigial esterase domain but showed no obvious hemagglutination inhibition activity; instead, it interfered with release of progeny virions from infected cells (58).…”

A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

“…A cell fusion inhibition assay was performed as described previously (31). In brief, Vero cells were grown to 80% confluence in 24-well plates and transfected with plasmid pcDNA-H5 (1.6 mg of total DNA per well) with Lipofectamine 2000 (Invitrogen) according to the instruction manual.…”

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

“…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)(5)(6)(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, Y168, and T171 according to H3 numbering) (9) (Fig. 1H), and mouse MAb NR2728 at S121 (S125 according to H3 numbering) (10) (Fig.…”