Abstract

Inactivated vaccines are widely used in fish disease control, but their limited immunogenicity restricts their protective efficacy. In this study, grass carp CXCL20a was optimized by site-directed mutagenesis, and an S35R mutant (M-C20) was evaluated as a molecular adjuvant for an inactivated grass carp reovirus (GCRV) vaccine. Structural prediction suggested that the S35R substitution may enhance the interaction between CXCL20a and its receptor CXCR3.1b1, which was further supported by pull-down analysis. Compared with wild-type C20, M-C20 showed stronger chemotactic and phagocytosis-promoting activities. Both C20 and M-C20 enhanced the efficacy of the inactivated vaccine after GCRV-II challenge. The final survival rate increased from 56% in the vaccine-alone group to 72% and 80% in the Va + C20 and Va + M-C20 groups, respectively. The relatively better protection observed in the Va + M-C20 group was further supported by lower viral load, milder histopathological damage, more favorable serum enzyme profiles, including lower LDH activity and relatively higher ACP and SOD activities, and higher expression of immune-related genes, including TNF-α, MHCII β, Gig1, and IgM, in the spleen and head kidney. These results indicate that both C20 and M-C20 improved the protective efficacy of the inactivated GCRV vaccine in grass carp, whereas M-C20 showed a relatively stronger adjuvant effect.