Multi-Epitope Vaccine Against African Swine Fever Virus Designed Through Reverse Vaccinology and Genomic Analysis with Experimental Validation of Immunogenicity

African swine fever (ASF), a highly pathogenic and lethal disease caused by the African swine fever virus (ASFV), poses a critical threat to global swine production, with no licensed vaccines currently available. This study employed whole-genome analysis of representative ASFV strains (genotypes I, II and recombinant variants) combined with reverse vaccinology to screen linear B-lymphocyte (LBL), cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes for multi-epitope vaccine development. The designed vaccine comprised 36 antigenic epitopes derived from 18 consensus proteins across six representative ASFV strains, demonstrating high immunogenicity, non-toxicity, non-allergenicity and favorable physicochemical stability. Molecular docking revealed strong binding affinity between the vaccine candidate and Toll-like receptor 4 (TLR4). Immunization experiments in mice demonstrated significant induction of specific antibodies alongside robust humoral and cellular immune responses. These findings underscore the vaccine’s potential for comprehensive prevention against diverse ASFV strains, providing a pivotal foundation for advancing ASF vaccine development.