Antibody 3CF5 neutralizes historical and emerging H5Nx influenza viruses by targeting a conserved non-receptor-binding hemagglutinin epitope

Avian influenza viruses of the H5Nx subtype pose persistent pandemic threats. Here, we immunized rhesus macaques with an H5N6 vaccine and obtained 30 H5-reactive monoclonal antibodies (mAbs). Three IgG mAbs, 3CF5, 3BG3, and 3BE7, demonstrated potent neutralizing activity against multiple H5Nx viruses and conferred protection in a lethal mouse challenge model. Notably, 3CF5 exhibited broad neutralization against H5Nx viruses spanning 1997-2024, including contemporary clade 2.3.4.4b H5N1 strains associated with an ongoing outbreak in dairy cattle. Cryo-electron microscopy (cryo-EM) analysis revealed that 3CF5 targets a conserved non-receptor-binding epitope on the hemagglutinin (HA) head, spanning the vestigial esterase domain and an adjacent lateral surface. 3CF5 mediates neutralization primarily through inhibiting membrane fusion and also possesses antibody-dependent cellular cytotoxicity. Furthermore, isotype conversion of 3CF5-IgG to mucosal secretory IgA1 (sIgA1) enhanced neutralization potency against H5N1 viruses up to 128-fold, highlighting the advantage of sIgA1 as a nasally administered prophylactic agent.