Immunopotentiating effects of recombinant TNFα in formalin-killed Streptococcus parauberis vaccine for starry...

To enhance vaccine efficacy in aquaculture species, molecular adjuvants such as cytokines are gaining attention for their immunostimulatory potential. In this study, we evaluated the functional role of recombinant tumor necrosis factor-alpha (rTNFα) as a molecular adjuvant in starry flounder (Platichthys stellatus) vaccinated against Streptococcus parauberis. Intraperitoneal administration of rTNFα at different concentrations (10-100 μg/mL) significantly induced pro-inflammatory cytokines (IL-1β, TNFα), apoptotic regulators (Caspase-3, Caspase-8), and cytolytic effectors (Perforin-2), in a dose- and time-dependent manner. Notably, STAT3 expression peaked at 12 h, and Perforin-2 exhibited a ∼400-fold increase by day 7, indicating sustained immune activation. In vivo, FKC vaccination alone conferred dose-dependent protection, but co-administration with rTNFα further improved survival (up to 100 %) and maintained high antigen-specific IgM titers through 6 weeks. These results confirm that rTNFα promotes both innate and adaptive immune responses and significantly enhances the protective efficacy of the FKC vaccine. Our findings highlight rTNFα as a promising molecular adjuvant for developing next-generation vaccines in finfish aquaculture.