Impact of naturally occurring hemagglutinin substitutions on antigenicity and fitness of influenza A(H5N1) virus.

In 2024, a human infection with clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) virus was identified in the United States in an individual with no known exposure. Genetic analysis revealed two hemagglutinin (HA) substitutions, P136S and A156T, which may alter viral antigenicity. Virus isolation was unsuccessful, preventing timely serologic analysis. To overcome this limitation, we generated recombinant viruses by reverse genetics and characterized the effects of the substitutions on antigenicity, receptor binding, and replicative fitness. The A156T substitution introduced a potential N-linked glycosylation site, resulting in altered antigenicity and reduced replication in primary human nasal epithelial cells and ferrets. Importantly, the A(H5N1) candidate vaccine virus (CVV) IDCDC-RG80A, which possesses HA-T156, remained antigenically effective against viruses with and without these substitutions. These findings highlight the importance of sequencing, reverse-genetics approaches, and antigenically similar CVVs such as IDCDC-RG80A, for pandemic preparedness against evolving clade 2.3.4.4b A(H5N1) viruses.