Unlocking Immune Signatures: Surrogate Markers for Assessing VHSV Vaccine Efficacy in Olive Flounder (Paralichthys olivaceus)

<i>Viral hemorrhagic septicemia virus</i> (VHSV) is a major pathogen in <i>Paralichthys olivaceus</i> (olive flounder) aquaculture, necessitating effective vaccines and evaluation methods. This study assessed immune responses following intraperitoneal administration of an inactivated VHSV vaccine and identified surrogate markers of vaccine efficacy. Fish were sampled weekly over 8 weeks. Antigen-specific antibody titers were measured by ELISA, and the expression of seven immune-related genes (<i>CD4</i>, <i>CD8</i>, <i>CD28</i>, <i>IgM</i>, <i>Mx</i>, <i>IFN</i>, and <i>IL-1β</i>) was analyzed in the spleen, kidney, liver, and gill by qPCR. The vaccinated group showed 84% relative survival, while unvaccinated fish experienced 100% mortality. Antibody titers peaked at 4 weeks post-vaccination, aligning with elevated <i>IgM</i> expression in the spleen and kidney. <i>CD4</i> (kidney) and <i>CD28</i> (spleen) expression strongly correlated with antibody titers (r = 0.854 and 0.796, respectively), whereas <i>IL-1β</i>, <i>IFN</i>, and <i>Mx</i> showed moderate associations in specific tissues. These findings indicate that <i>CD4, CD28</i>, and <i>IgM</i> may serve as molecular indicators of humoral immune responses specifically following inactivated VHSV vaccination in olive flounder (<i>Paralichthys olivaceus</i>). Combining gene expression profiling with antibody analysis offers a non-lethal, practical approach to evaluating vaccine efficacy in aquaculture.