Oral immunizations with Bacillus subtilis spores expressing MCP protein provide protection against red-spotted grouper nervous necrosis virus (RGNNV) infection in juvenile grouper, Epinephelus coioides

Abstract

Nervous necrosis virus (NNV) causes severe viral disease in grouper, resulting in major economic losses in the global grouper aquaculture industry as a result of the high mortality rate. In the present study, we aimed to develop a safe and efficient vaccine to protect grouper against NNV infection. To achieve effective oral and bath immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as the vehicle to deliver MCP (major capsid protein of RGNNV) partnered with CotC, one of the coat proteins of B. subtilis, to the gastrointestinal tract. After routine culturing for 8–12 h in LB medium, B. subtilis containing CotC-MCP (B.s-CotC-MCP) was transferred into the sporulation culture medium. Western blot analysis and the growth curve indicated that the best sporulation time for recombinant WB600 was 24–30 h at 37 °C with continuous shaking (250 rpm). The grouper then received surface displaying B·S-CotC-MCP vaccine against RGNNV via either oral or bath immunization and their immune responses were monitored. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly enhanced in juvenile grouper following oral or bath immunizations (1 × 106, 1 × 107, and 1 × 108 CFU g−1). The levels of specific antibodies against RGNNV and the transcription of immune-related genes (TNF-α, IL-1β, MHCI and IgM) were also significantly enhanced in juvenile grouper flowing oral or bath immunizations (1 × 106, 1 × 107, and 1 × 108 CFU g−1). However, only the highest dose of bath vaccination induced the production of specific antibodies significantly and up-regulated transcriptions of several immune-related genes (IgM and MHCI). The lower cumulative mortality in vaccinated groups after RGNNV challenge clearly demonstrated that surface-displayed MCP vaccine could protect fish against RGNNV infection. The relative percentage survival (RPS) value in the orally vaccinated group (88.89%) was much higher than the bath group (18.89%), which was consistent with the production of specific serum antibodies, non-specific immune response and immune related gene expression. Therefore, this study demonstrated that the CotC-MCP spores can trigger high levels of mucosal and humoral immunity, and represent a promising candidate vaccine against NNV infection.