Currently, immunization with inactivated influenza virus vaccines is the most prevalent method to prevent infections. However, licensed influenza vaccines provide only strain-specific protection and need to be updated and administered yearly; thus, new vaccines that provide broad protection against multiple influenza subtypes are required. In this study, we demonstrated that intradermal immunization with gp96-adjuvanted seasonal influenza monovalent H1N1 split vaccine could induce cross-protection against both group 1 and group 2 influenza A viruses in BALB/c mice models. Vaccination in the presence of gp96 induced an apparently stronger antigen-specific T cell response than split vaccine alone. Immunization with the gp96-adjuvanted vaccine also elicited apparent cross-reactive CD8+ T cell response that targeted the conserved epitopes across different influenza virus strains. These cross-reactive CD8+ T cells might be recalled from a pool of memory cells established after vaccination and recruited from extra-pulmonary sites to facilitate viral clearance. Of note, six highly conserved CD8+ T epitopes from the viral structural proteins HA, M1, NP and PB1 were identified to play a synergistic role in gp96-mediated cross-protection. Comparative analysis showed that most of conservative epitope-specific CTLs apparently induced by heterologous virus infection were also activated by gp96-adjuvanted vaccine, thus resulting in broader protective CD8+ T cell responses. Our results demonstrated the advantage of adding gp96 to an existing seasonal influenza vaccine to improve its ability to provide better cross-protection. Importance Owing to continuous mutations in hemagglutinin (HA) or neuraminidase (NA) or recombination of the gene segments between different strains, influenza viruses can escape the immune responses developed by vaccination. Thus, new strategies aimed to efficiently activate immune response that targets to conserved regions among different influenza viruses are urgently needed in designing broad-spectrum influenza vaccine. Heat shock protein gp96 is currently the only natural T cell adjuvant with special ability to cross-present coupled antigen to MHC I molecule and activate downstream antigen-specific CTLs response. In this study we demonstrated the advantages of adding gp96 to monovalent split influenza virus vaccine to improve its ability to provide cross-protection in BALB/c mice model and proved that gp96 activated cross-reactive CTL response is indispensable in our vaccine strategy. Due to its unique adjuvant properties, gp96 might be a promising adjuvant for designing new broad-spectrum influenza vaccines.

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