Intranasal vaccine induces broad and long-lasting immunity against the hemagglutinin stem of group 2 influenza A viruses

Influenza A viruses are categorized into two phylogenetic groups (group 1 and group 2) based on the structure of their hemagglutinin (HA) protein. Within group 2, H3N2 poses a particular challenge due to its rapid evolution, limited vaccine efficacy, and association with more severe influenza seasons. Although T cell responses have been extensively studied in the context of vaccine-induced protection, HA stem (HA2)-specific T cell responses have been relatively understudied, especially those related to nasal immunity. To address this, we engineered an adenoviral vector vaccine (Ad-HA2) expressing a consensus hemagglutinin stem sequence, derived through bioinformatic analysis of all H3 strains. The vaccine conferred heterosubtypic protection against lethal challenges with either H3N2 or H7N9, both belonging to group 2 influenza A viruses, with protection lasting at least six months post-vaccination. Notably, the vaccine induced robust HA2-specific humoral and cell-mediated responses in the nasal-associated lymphoid tissue (NALT) of the upper respiratory tract, the first line of immune defense against inhaled pathogens. The vaccine also elicited significant levels of antibodies and T cell responses in the lower respiratory tract and pulmonary immune sites. Furthermore, circulating antibodies in the serum demonstrated effective antibody-dependent cellular cytotoxicity (ADCC) activity. Finally, using a peptide pool matrix screening approach combined with in silico verification, we identified an immunogenic C-terminus region of the HA2 consensus sequence that activated CD4⁺ and CD8⁺ T cells, which warrants further investigation. Collectively, these findings are informative for the design and evaluation of mucosal influenza vaccines targeting the hemagglutinin stem.