An H5N1 clade 2.3.4.4b virus vaccine that elicits cross-protective antibodies against conserved domains of H5 and N1 glycoproteins

The continuous evolution and global spread of highly pathogenic avian influenza (HPAI) H5N1 viruses, particularly clade 2.3.4.4b, pose major challenges for pandemic preparedness. This study evaluates a low-dose inactivated split-virus vaccine derived from H5N1 clade 2.3.4.4b, formulated with an Alum/CpG adjuvant, in a preclinical female mouse model. The vaccine induces strong humoral and cellular immunity, generating high titers of cross-reactive antibodies against diverse H5 hemagglutinin (HA) and across different N1 neuraminidase (NA) glycoproteins. The Alum/CpG adjuvant supports substantial antigen dose sparing and promotes a balanced Th1/Th2 profile. Functional assays show potent virus neutralization, neuraminidase inhibition, and antibody-dependent cellular cytotoxicity, alongside robust antigen-specific CD4⁺ and CD8⁺ T cell responses, efficient control of lung viral replication, and reduced lung inflammation. Vaccinated mice are fully protected from lethal challenge with both homologous H5N1 clade 2.3.4.4b and heterologous clade 1 viruses, despite low hemagglutination inhibition (HAI) titers. Electron microscopy polyclonal epitope mapping shows serum antibodies recognizing multiple epitopes on homologous HA and NA, with cross-reactivity to conserved epitopes on heterologous proteins, indicating broad recognition. Together, these findings support this vaccine candidate as a promising strategy to provide broad, multifunctional, and durable immunity against current and emerging H5N1 threats.