Construction and immunogenicity evaluation of recombinant Bacillus subtilis expressing VP8 protein of porcine rotavirus

Abstract

Porcine rotavirus (PoRV), an enteric infectious virus, is a major cause of neonatal diarrhea in piglets, resulting in significant economic losses to the global swine industry. In this study, the cholera toxin B subunit (CTB) was utilized as a molecular adjuvant to construct recombinant Bacillus subtilis ( B. subtilis ) strains expressing VP8 proteins and CTB-VP8 fusion proteins derived from the PoRV genotypes P[7], P[13], and P[23]. The recombinant strains expressing PoRV proteins from different genotypes were combined and orally administered to mice, and their immunogenicity and probiotic functions were evaluated. The results demonstrated that the recombinant B. subtilis significantly increased serum-specific IgG and fecal-specific sIgA antibodies levels ( P  < 0.001), indicating that the recombinant strains induced both humoral and mucosal antibody responses in mice. Notably, the CTB-fused strains elicited higher titers of IgG and sIgA. Furthermore, the recombinant strains significantly elevated the expression levels of cytokines, including IFN-γ, IL-2, and IL-4, in both serum and intestinal mucosa ( P  < 0.01), suggesting a Th1/Th2-biased immune response at the cytokine level. In addition, administration of recombinant B. subtilis increased villus height and the villus height-to-crypt depth ratio while reducing crypt depth, showing a tendency to improve intestinal morphology. This study addresses the limitations associated with insufficient mucosal immune activation by conventional injectable approaches and the absence of cross-protection among different genotypes of PoRV, thereby providing a theoretical foundation for the development of an oral trivalent live-vector vaccine.