In the aquaculture industry, disease control remains a significant concern, with vaccination being a pivotal strategy due to its cost-effectiveness and long-lasting protection. However, limited understanding of fish immunology has hindered the development of effective vaccines, often relying on trial-and-error methods. This study aimed to investigate immune-related gene expression during the vibrio vaccination incubation period, elucidating key immune pathways in orange spotted grouper (Epinephelus coioides). By comparing survival rates in challenge trials and identifying highly expressed genes associated with vaccine protection, we found distinct differences in the expression of immune-related genes, notably IL-6 and TNF-γ.
Subsequent administration of recombinant IL-6 and TNF-γ proteins revealed their ability to induce CD4+ and CD8+ lymphocyte proliferation via the Th2 and Th1 pathways, respectively. Further experiments involved adding these proteins to vibrio vaccine contain commercial adjuvant Seppic MontanideTM ISA 763A to enhance immune effects. Results showed that IL-6 addition surpassed commercial adjuvants alone, and surpassed TNF-γ addition in specific antibody potency, while TNF-γ addition outperformed IL-6 in terms of protection, and better than commercial adjuvants alone. These findings underscore the importance of modulating Th1 and Th2 lymphocyte proliferation for vaccine efficacy. Incorporating these immune molecules into vaccines significantly improved survival rates in pathogen challenge tests, offering insights into fish immune responses and suggesting a promising approach for enhancing vaccine efficacy.