An improved oral vaccine with molecular adjuvant ?-defensin protects grouper against nervous necrosis virus infection

Nervous necrosis virus (NNV) is one of the most important fish viral pathogens infecting more than 120 fish species worldwide. Due to the mass mortality rates often seen among larvae and juveniles, few effective vaccines against NNV were developed up to now. Here, the protective effect of recombinant coat protein (CP) from red -spotted grouper nervous necrosis virus (RGNNV) fused with grouper beta-defensin (DEFB) as an oral vaccine was evaluated using Artemia as a biocarrier delivery system in pearl gentian grouper (Epinephelus lanceolatus male x Epinephelus fuscoguttatus female). Feeding with Artemia encapsulated with E. coli expressing control vector (control group), CP, or CP-DEFB showed no obvious side effects on the growth of groupers. ELISA and antibody neutralization assay showed that CP-DEFB oral vaccination group induced higher anti-RGNNV CP specific an-tibodies and exhibited higher neutralization potency than the CP and control group. Meanwhile, the expression levels of several immune and inflammatory factors in the spleen and kidney after feeding with CP-DEFB were also significantly increased compared with the CP group. Consistently, after challenge with RGNNV, groupers fed CP-DEFB and CP exhibited 100% and 88.23% relative percentage survival (RPS), respectively. Moreover, the lower transcription levels of viral genes and milder pathological changes in CP-DEFB group were detected compared with the CP and control group. Thus, we proposed that grouper beta-defensin functioned as an efficient molecular adjuvant for an improved oral vaccine against nervous necrosis virus infection.