Immune and protective effects of recombinant multi-epitopes vaccine against infectious spleen and kidney necrosis virus in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus).

The capsid protein (CP), the primary structural component and key antigen of infectious spleen kidney necrosis virus (ISKNV), remains poorly characterized in terms of its antigenic epitopes. These epitopes, typically ∼20 amino acid residues in length, are specific chemical groups determining antigenic specificity. To address this gap, we employed immunoinformatics to analyze CP epitopes. Subsequently, a multi-epitope vaccine targeting ISKNV (MEV) was designed, constructed, and screened. Immune response evaluation revealed that the predicted three T lymphocyte and five B lymphocyte epitopes exhibited antigenicity, non-toxicity, and non-allergenicity. Fusion of these epitopes yielded the MEV vaccine. In grouper trials, MEV vaccination significantly upregulated mRNA expression of immune-related genes (IL-1β, TNF-α, CD4, CD8α, MHC-Iα, MHC-IIα) and markedly increased total IgM levels, indicating induction of both cellular and humoral immunity. Crucially, MEV conferred significantly higher resistance against ISKNV challenge compared to CP immunization, achieving 85.33% survival rate. Furthermore, MEV vaccination significantly reduced viral loads in the spleen and head kidney. This study presents an economical, rapid, safe, and effective strategy for aquatic vaccine development, positioning MEV as a highly promising candidate for preventing infectious spleen kidney necrosis disease.