Abstract

Successful construction of Bacillus subtilis spores expressing CSBV N protein. • Deletion of the CotC stop codon enables stable surface display of CSBV N fusion protein in recombinant spores. • Recombinant spores significantly triggered immunity of Japanese flounder. Chinook salmon bafinivirus (CSBV) poses a considerable threat to the aquaculture of Japanese flounder ( Paralichthys olivaceus ), resulting in substantial economic losses globally. Oral vaccination, using Bacillus subtilis spore surface display for antigen delivery, offers a promising approach due to its low stress and scalability. In this study, two potential oral vaccine candidates based on recombinant spores, B.s -CotC(TAA)-CSBV N and B.s -CotC(△TAA)-CSBV N, were constructed by fusing the CSBV nucleoprotein (N) gene with the CotC anchor gene, either with or without the stop codon. Successful surface display of the CSBV N protein was confirmed in the B.s -CotC(△TAA)-CSBV N group. Oral immunization revealed that removal of the CotC stop codon was essential for full-length expression and stable anchoring of the fusion protein, significantly enhancing IgM transcription and specific antibody levels. The recombinant spores crossed the gastrointestinal barrier and targeted immune tissues, including the intestine, spleen, and kidney, with predominant localization in the intestinal mucosa, splenic marginal zone, and renal glomeruli. The resulting immune response was suboptimal, a finding potentially linked to the restricted passage of antigen into the white pulp of the spleen. The spores remained stable at 4 °C, retaining about 60% immunoreactivity after four weeks. Overall, these results indicated that the CotC stop codon-deleted B. subtilis spore system is a promising oral vaccine candidate against CSBV, effectively stimulating systemic immunity in P. olivaceus and offering a useful strategy for managing viral diseases in aquaculture.