A mosaic H3 subtype live attenuated influenza vaccine elicits broad immune responses to influenza a viruses

Influenza A(H3N2) viruses are typically associated with reduced vaccine effectiveness (VE), especially in the elderly. To address this challenge, we developed a novel live attenuated influenza vaccine (LAIV) expressing a mosaic H3 hemagglutinin designed to maximize potential cytotoxic T lymphocyte (CTL) epitope coverage, using the A/Leningrad/134/17/57 (H2N2) backbone. The resulting mosaic reassortant virus exhibited temperature-sensitive and cold-adapted phenotypes and caused no significant weight loss or clinical signs in mice. Following prime-boost intranasal immunization in BALB/c mice, robust cellular immune responses were observed, with increased frequencies of splenic and pulmonary IFN-γ⁺ and IL-4⁺ T cells after restimulation, as well as elevated levels of pulmonary tissue-resident memory T cells. High levels of cross-reactive IgA were also detected in nasal and bronchoalveolar lavage fluids, covering multiple influenza virus strains. Additionally, broad serum antibody responses were induced, with HI and MN antibodies targeting 8 and 7 of 9 H3N2 vaccine strains. Virus challenge results showed that the mosaic reassortant virus conferred complete protection against the homologous-subtype strain A/Aichi/2/1968 (H3N2) and partial protection against the heterosubtypic strains A/Puerto Rico/8/1934 (H1N1) and A/Hunan/42443/2015 (H1N1), with survival rates of 20 % and 40 %, respectively. In contrast, all mice vaccinated with traditional monovalent LAIV or inactivated influenza vaccine showed 0 % survival. Moreover, lung viral loads showed a decreasing trend after infection, and pathological damage in the lungs was markedly alleviated compared to other groups. These findings suggest that combining the mosaic antigen with LAIV induces multi-layered immune responses and provides a rational vaccine design strategy for addressing the rapid antigenic evolution of H3N2 viruses.