A Panel of Human Monoclonal Antibodies for Tracking the Antigenic Evolution of Influenza H5N1 Clade 2.3.4.4b

The ongoing panzootic of clade 2.3.4.4b H5N1 influenza has resulted in widespread infection of birds, mammals, and livestock, underscoring the need for tools to interpret its real-time evolution. Here, we describe the isolation and characterization of a panel of 19 human monoclonal antibodies that potently neutralize current isolates. Competition immunoassays and cryo-electron microscopy analyses revealed their collective near-complete epitope coverage of the H5-hemagglutinin surface. Neutralization profiling across multiple historical and contemporary H5 viruses defined their epitope-specific patterns of virus neutralization. One cluster of antibodies potently neutralized only clade 2.3.4.4b viruses, while many others exhibited broadly neutralizing activity against diverse H5N1 clades. Application of this structurally calibrated antibody panel to recent North American human isolates revealed genotype-specific antigenic divergence between lineages that have spread among cattle (B3.13) and poultry (D1.1). Together, the findings of this study establish a structurally grounded antibody reference panel spanning major vulnerable sites of H5 hemagglutinin and provide a toolbox for interpreting emergent mutations, monitoring ongoing antigenic drift, and anticipating the evolutionary trajectory of circulating H5N1 influenza viruses.