Multi-route induction of Salmonella-based vaccine delivery elicits protective immunity against FV3-like ranavirus in an ornamental fish model

Viruses belonging to the genus Ranavirus affect the ectothermic animal community worldwide. In this study, we employed a bioengineered Salmonella mutant, JOL2500, to deliver major capsid (MCP) and outer membrane proteins of frog virus 3 (FV3) to elicit FV3 specific immunity. The optimized antigen protein sequences were cloned into a prokaryotic expression vector, pJHL65. The recombinant plasmids were transformed into JOL2500, and the constructed strains were designated as JOL3171 and JOL3172 carrying MCP and membrane protein retrospectively. The constructed vaccine strains were administrated to an ornamental fish model via IP injection and immersion. The results demonstrated higher neutralizing antibody titers in JOL3171 immunized fish via both IP injection and immersion. At FV3 virus challenge, the JOL3171 vaccinated group exhibited 100 % survival and significantly decreased viral loads; other groups had mortality rates of 16.6–33.3 % and higher viral loads. The cytokine profiles indicated that JOL3171 vaccination via immersion induced distinct cytokine expression. Specifically, the group exhibited elevated levels of IFN-γ, TNF-α, and IL-16, indicative of a potent Th1-polarized cell-mediated immune response crucial for MHC-I restricted viral clearance. Importantly, this pro-inflammatory surge was balanced by upregulated IL-10. Immunohistochemistry showed that intestine and epithelial tissue is the main invasion route of immersion-administered Salmonella. In conclusion, our results provides preliminary evidence that Salmonella vaccine delivering major capsid protein via immersion can induce FV3-specific immunity causing rapid clearance and neutralizing antibody production.