Immunoreactivity of pan MHC-I epitopes on Crimean-Congo hemorrhagic fever virus glycoprotein C-terminal.

Introduction: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne pathogen causing severe hemorrhagic disease with high mortality. The viral glycoprotein Gc mediates membrane fusion and represents a key target of CD8⁺ T-cell responses. However, systematic identification and comprehensive evaluation of pan-MHC-I-restricted Gc epitopes remain limited. Methods: An integrated immunoinformatics workflow incorporating five prediction algorithms (IEDB, NetMHCpan4.1, SMMPMBEC, SYFPEITHI, and Rankpep) was applied to identify high-affinity 9-mer epitopes from the CCHFV Gc protein restricted by human HLA-I and murine H2 alleles. Immunogenicity, conservation, toxicity, and allergenicity were assessed using established computational tools. Peptide-MHC interactions were further examined by molecular docking and molecular dynamics simulations. Selected epitopes were experimentally validated by ELISpot assays in BALB/c and SJL mice immunized with a Gc-based DNA vaccine. Results: Ninety-four human HLA-I and thirty-seven murine H2-restricted dominant epitopes were predicted. Among these, 21 epitopes exhibited high binding affinity, favorable immunogenicity, and broad conservation across viral strains. Most candidates showed low predicted toxicity and allergenicity. Structural analyses supported stable peptide-MHC interactions. ELISpot assays confirmed that several epitopes, including APFILLILF and FVKWKVEYI, elicited significant IL-2 responses, indicating functional T-cell activation. Conclusion: This study provides a systematic framework for identifying conserved and immunogenic pan-MHC-I-restricted epitopes within the CCHFV Gc protein. The validated candidates may support the rational design of multi-epitope vaccines and contribute to understanding cellular immune responses against CCHFV.