Diminished sialoside binding in novel H5N1 influenza hemagglutinin variants identified in a human patient

In 2024, an adolescent female in British Columbia was hospitalised presenting with severe symptoms including respiratory failure due to infection with a novel H5N1 influenza strain (BC24). Using cryogenic electron microscopy, we show here that the N169 α2,3-linked auto-glycan that is found in the sialic acid binding site of previously studied H5 hemagglutinin (HA) proteins is absent in purified BC24 HA protein, suggesting greatly reduced affinity for α2,3-linked sialosides. Glycan microarray and enzyme-linked immunosorbent assay analyses show that HA variants identified in the BC24 case display severely reduced or no binding to both α2,3-linked sialosides and α2,6-linked sialosides. Full-length BC24 HA expressed in A549 alveolar carcinoma cells drives membrane fusion, albeit at lower levels than previous H5 HA proteins, and post-infection sera from the patient display strong binding to BC24 HA and HA proteins from other influenza subtypes. As each of the two mutations of interest, independently and in conjunction, severely reduce sialoside binding, there appears to have been in this case multiple populations of virus with the diminished receptor binding phenotype. The substantial minority prevalence of weakly binding HA variants in this BC24 case reveals further complexity in the factors that may be present in severe avian influenza infection. This study explores the biochemical and structural roles of hemagglutinin mutations identified in a human case of H5N1 influenza and reports effects of the mutations in patient viral populations on sugar binding and membrane fusion.