Abstract
The high mutation rate of the influenza virus poses a significant challenge to global health, highlighting the urgent need for broad-spectrum vaccines. Here, we engineered Ad-Hex, a pan-influenza vaccine comprising three chimpanzee adenoviral vectors (Ad-H1H3, Ad-BYBV, and Ad-H5H7), each encoding the hemagglutinin (HA) genes from two distinct influenza virus subtypes/lineages in the △E1 region. A single intranasal dose of Ad-Hex induced robust humoral, cellular, and mucosal immunity, conferring complete protection against lethal challenge with six vaccine-matched strains in female C57BL/6 or Balb/c mice. Notably, the vaccine achieved 60% survival rates against mismatched strains. Mechanistic studies revealed that Ad-Hex activated germinal center B-cell responses not only against the cognate HAs but also against the conserved HA stalks. This drove the production of cross-reactive antibodies, which contributed to heterologous protection along with the CD8(+) T cell response. Our study confirms the feasibility of this multivalent strategy by mixing multiple recombinant adenoviruses and provides insights into the development of multivalent vaccines against other respiratory pathogens.