Optimization, Physicochemical Characterization, and Immunoprotective Efficacy of an Oral Inactivated Largemouth Bass Ranavirus Vaccine Encapsulated in N,O-Carboxymethyl Chitosan-Chitosan Nanoparticles.

Viral diseases pose a significant threat to aquatic animal health, and no effective treatment is currently available. Vaccines represent the most efficient approach to preventing viral infections. Because of the aquatic habitat, injection-based vaccination is time-consuming and labor-intensive, whereas oral vaccines lack a suitable delivery system. This study aimed to prepare N,O-Carboxymethyl Chitosan (NOCMS)-Chitosan (CS) nanoparticles (NOCMS-CS NPs) via a self-assembly method for the delivery of inactivated largemouth bass ranavirus (LMBV) vaccine (LMBV-iV). Single-factor experiments, the steepest ascent method, and response surface methodology were combined to optimize the preparation process. The optimal conditions were a CS concentration of 4.00 g/L, an NOCMS concentration of 2.86 g/L, an NOCMS:CS ratio of 1:1.66, a vaccine:carrier ratio of 1:4.43, and 2% glacial acetic acid, yielding an encapsulation efficiency of 77.31%. Following oral administration of LMBV-iV delivered by NOCMS-CS NPs, the relative percent survival (RPS) of largemouth bass after LMBV infection reached 65%. Furthermore, the carrier-delivered vaccine reduced the viral load in target organs and significantly increased serum IgM, mucosal IgT in the mid-hind intestine, immune-related enzyme activities (AKP, ACP, LZM, SOD) in the intestinal mucus, and the expression of immune-associated genes (IgM, IgT, TNF-α, IL-1β, IL-8, IFN-γ) in the head kidney and spleen. In conclusion, this NOCMS-CS NPs carrier showed considerable potential as an oral vaccine delivery system for aquaculture.