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.
Highlights
Pathogenic avian influenza (HPAI) H5N1 viruses continue to spread among poultry and have frequently broken the species barrier and transmitted to humans
Previous studies concluded that human monoclonal antibody (mAb) AVFluIgG01 targets a linear epitope within a sequence of the 116IIPKSSWSS124 in the global head region of HA [12]
This result implied that the epitope for AVFluIgG01 could not be located within the 116IIPKSSWSS124 motif
Summary
Pathogenic avian influenza (HPAI) H5N1 viruses continue to spread among poultry and have frequently broken the species barrier and transmitted to humans. The viral hemaglutinin (HA) surface glycoprotein of influenza A viruses is responsible for binding to cell receptor and a primary target of neutralizing antibodies. It is initially synthesized as a precursor polypeptide (HA0) and subsequently cleaved by cellular proteases into disulfide-linked HA1 and HA2 subunits. The H3 structure was used to map the antigenic sites of H1 [2], H2 [3], and H5 [4] subtypes. Kaverin et al [8] demonstrated that the HA antigenic structure of recent HPAI H5N1 isolates differs substantially from that of low-pathogenicity H5 strains described earlier and is rapidly evolving
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