An immunoinformatics-derived multi-epitope DNA vaccine targeting the Micropterus salmoides rhabdovirus G protein elicits protective immunity in largemouth bass

Largemouth bass (Micropterus salmoides) is a commercially important freshwater aquaculture species. However, the sustainable development of this industry is severely threatened by Micropterus salmoides rhabdovirus (MSRV), a critical pathogen that causes acute mortality and substantial economic losses. In this study, five cytotoxic T lymphocyte (CTL) epitopes, three helper T lymphocyte (HTL) epitopes, and two B cell epitopes were identified from the MSRV G protein using immunoinformatics approaches. By serially linking these epitopes, a multi-epitope antigen (Multi-G) consisting of 149 amino acids was further constructed. The Multi-G was cloned into pEGFP-N1 to generate the recombinant DNA vaccine pEGFP-Multi-G. Both single and double immunization with pEGFP-Multi-G induced Multi-G-specific antibodies in fish serum. Importantly, the DNA vaccine provided significant protection against MSRV infection in largemouth bass, with relative percent survival (RPS) values of 56.7% in the single immunization group and 63.3% in the double immunization group. Furthermore, pEGFP-Multi-G immunization inhibited MSRV replication and alleviated histopathological lesions in the spleen, liver, and hindgut. Collectively, pEGFP-Multi-G elicits specific immune responses in largemouth bass, laying a solid theoretical and experimental foundation for the development of multi-epitope DNA vaccines against MSRV.