T-cell epitope supplementation enhanced cross-protection against H7N9 AIV in chickens via upregulated HA epitope-specific T cell and CD4 T cell-mediated help

• Four conserved T-cell epitopes recognized by chicken CD4 + and/or CD8 + T cells were identified within H7N9 HA protein • An H7N9 WIV nanoparticle vaccine supplemented with the four T-cell epitopes (H7N9 WIV + PP) elicited stronger antibody and T-cell responses than WIV alone. • The H7N9 WIV + PP vaccine induced cross-protection against antigenically divergent H7N9 challenge in poultry. • The enhanced cross-protection against H7N9 AIV in chickens is related to upregulated HA epitope-specific T cells and CD4 T cell-mediated help. The continuous circulation of H7N9 avian influenza viruses (AIVs) in poultry poses a significant public health threat. T cell-mediated immunity favors enhanced and broad protection against viruses. However, it remains challenging to exploit avian T-cell immunity for improving vaccine efficacy. Here, we showed that vaccine-induced hemagglutinin (HA)-specific T-cell responses promoted broad protection against heterologous H7N9 in poultry. Four conserved epitopes of H7N9 HA recognized by chicken T cells were identified, including two CD4 T-cell epitopes (P12, P15), one CD8 T-cell epitope (P4), and one dual-recognized epitope peptide (P10). These peptides were pooled (PP) and incorporated into an H7N9 whole inactivated virus (WIV) vaccine via a DDA/TDB nanoparticle platform (H7N9 WIV + PP) for better delivery and presentation of diverse T-cell epitopes. The results showed that H7N9 WIV + PP vaccination elicited more robust humoral and cellular immune responses than the H7N9 WIV alone and conferred broad protection against antigenically divergent H7N9 virus challenge. Mechanistically, pooled T-cell epitope supplementation enhanced the elicitation of HA peptide-specific T-cell responses, which in turn boosted HA-specific antibodies via peptide-specific CD4 + T-cell help while concurrently mobilizing protective CD4 + and CD8 + T-cell immunity and effector molecules. Our study offers a novel strategy for developing broad-spectrum poultry vaccines against evolving AIV strains. Introduction: The persistent circulation of H7N9 avian influenza viruses (AIVs) in poultry continues to threaten public health. While T cell-mediated immunity is known to enhance and promote cross-protective antiviral responses, exploiting avian T-cell immunity for improved vaccine efficacy remains challenging. Hemagglutinin (HA)-specific T-cell responses may serve as a key mechanism to broaden protection against antigenically diverse H7N9 strains. Objectives: This study aimed to determine whether HA-specific T-cell responses can enhance heterologous protection against H7N9 in poultry and whether T-cell epitope-supplemented vaccines can improve both humoral and cellular immunity. Methods: Conserved HA epitopes recognized by chicken T cells were identified using immunological assays, resulting in two CD4 epitopes (P12, P15), one CD8 epitope (P4), and one dual-recognized epitope (P10). These peptides were pooled (PP) and incorporated into an H7N9 whole inactivated virus (WIV) vaccine formulated with a DDA/TDB nanoparticle adjuvant (H7N9 WIV + PP). Immune responses and protection were assessed following homologous and heterologous H7N9 virus challenge. Results: The H7N9 WIV + PP vaccine elicited stronger humoral and cellular immune responses compared with WIV alone. Birds receiving H7N9 WIV + PP displayed significantly improved protection against antigenically divergent H7N9 challenge. Mechanistically, pooled epitope supplementation enhanced HA peptide-specific T-cell responses, which subsequently augmented HA-specific antibody production via peptide-specific CD4 + T-cell help. Concurrently, both CD4 + and CD8 + T cells and their effector molecules were mobilized, contributing to broad protective immunity. Conclusion: Incorporating conserved HA-derived T-cell epitopes into an H7N9 WIV vaccine enhances both humoral and cellular immunity and confers broader protection against heterologous H7N9 strains. This strategy provides a promising approach for developing broad-spectrum poultry vaccines targeting continuously evolving AIVs.