Comparative immunobiology and cross-species validations of pan-MHC-II epitopes on Hantaan virus nucleocapsid protein

During viral infection, CD4⁺ helper T-cell is indispensable for the establishment of the humoral immune protection, CTL activation, and even long-term memory response. It requires MHC-II molecules in viral structural antigens process and presentation. HTNV NP epitopes exhibited high affinity to both of HLA-II superfamilies and H-2-I genes in the present study. Immunogenicity and conservation analyses identified 34 selective epitopes, later validated by molecular docking (MD) with MHC-II structures. NP 15-mer peptides and MHC-II haplotypes were found to interact bidirectionally through hierarchical clustering. In brief, epitopes that exhibit immunoreactivities for a wide range of MHC-II molecules reflect the biomedical practice of vaccination, while haplotype clusters reflect individual differences in T-cell antigen presentation. Then, 11 HTNV variants showed three amino acid substitutions in three epitopes, with little impact on their pan-HLA-II immunoreactivity. Safety analyses indicated that the 34 selective epitopes exhibit favorable safety profiles for potential applications. Finally, we validated the immunogenicity of the selective epitopes using ELISA, ELISpot, and flow cytometry. In conclusion, our work provides a comprehensive assessment of the pan-MHC-II immunoreactivity of HTNV NP and lays the theoretical and technical foundations for the development of protective epitope vaccines in the context of population immunity.