Surface-displayed multivalent PRRSV tandem antigen on <i>Bacillus subtilis</i> spores induces mucosal immune responses in mice

AIMS: Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to swine production, while safe and practical mucosal immunization strategies with broad reactivity are still limited. This study aimed to develop a spore surface-display mucosal immunomodulatory platform by presenting a multiepitope PRRSV antigen on Bacillus subtilis spores and to assess its ability to stimulate mucosal and systemic immune responses. METHODS AND RESULTS: An applied spore surface-display platform was constructed by engineering Bacillus subtilis 168 spores to present a tandem fusion antigen (ER) comprising conserved linear B- and T-cell epitopes derived from PRRSV ORF1b, GP5, M, and N proteins. Recombinant spores were administered orally to BALB/c mice as a proof-of-concept model, and mucosal and systemic immunogenicity was evaluated in comparison with control spores and a commercial inactivated vaccine. B. subtilis BE significantly increased ER-specific intestinal secretory IgA and serum IgG, and PRRSV (R98)-specific neutralising activity was detectable in the ER group. BE administration also promoted a Th1-skewed mucosal cytokine profile (increased IFN-α, TNF-α, and IFN-γ with reduced IL-6) and increased the proportion of CD8⁺ T cells in mesenteric lymph nodes. CONCLUSIONS: Surface display of a conserved multiepitope PRRSV antigen on B. subtilis spores stimulated coordinated mucosal, humoral, and cellular immune responses in mice. These findings support further exploration of spore-based microbial delivery systems as a potential mucosal immunisation adjuvant and immunomodulatory approach for PRRSV; however, the use of BALB/c mice represents a non-natural host model, and protective efficacy remains to be validated in target animals and challenge studies.