Immunoinformatics Design of a Broad-Spectrum Multi-Epitope Vaccine Targeting HA2 and M1 of H9N2 AIV.

H9N2 avian influenza virus (AIV) continues to mutate, leading to immunosuppression and secondary infections in poultry. Traditional inactivated vaccines mainly induce humoral immunity and have limited cross-protection efficacy against various subtypes of virus strains. In this study, we targeted the HA2 and M1 proteins of H9N2 as antigens and used immunoinformatics methods to design a broad-spectrum multi-epitope vaccine (MEV) that can simultaneously activate humoral and cellular immunity. Firstly, through systematic evolutionary analysis and sequence comparison, highly conserved amino acid sequence regions were selected from HA2 and M1 proteins. B-cell epitopes were predicted in the HA2 conserved sequence, and cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes were predicted in the M1 conserved sequence. Three candidate vaccines containing different epitope combinations were constructed. After secondary structure and physicochemical property comparisons, HM1 was determined as the optimal scheme. HM1 contains three B cell epitopes, two CTL epitopes, and three HTL epitopes, and was connected to chicken β-defensin at the N-terminus as a molecular adjuvant; a dendritic cell-targeting peptide was added at the C-terminus. The HM1 tertiary structure optimized by GalaxyRefine met the standards of a reliable model. The molecular docking results indicated that HM1 can form stable binding with chicken TLR2, TLR4, MHC I, and MHC II molecules, with binding free energies of -7.1 kcal/mol and -6.1 kcal/mol, respectively, and can form multiple hydrogen bonds and salt bridges. Normal mode analyses revealed that the HM1-TLR complex exhibits favorable dynamic properties at the computational level. The immune simulation prediction results showed that after vaccination with HM1, specific antibodies can be induced, B cells, helper T cells, and cytotoxic T cells can be activated, and IFN-γ and IL-2 can be secreted. In summary, the HM1 designed based on the conserved regions of HA2 and M1 proteins has good physicochemical stability and immunogenicity, providing a theoretical basis for the development of broad-spectrum and highly effective H9N2 vaccines.