Engineered Bacillus subtilis Spores Surface-Displaying a PRRSV GP3/GP4/GP5 Fusion Antigen: A Preliminary Immunogenicity Evaluation in Mice and Piglets.

Bacillus subtilis is a safe, non-pathogenic probiotic capable of forming highly resistant spores, making it an attractive chassis for oral antigen delivery and mucosal immune modulation. In this study, we engineered a recombinant B. subtilis strain, RG, to display an optimized PRRSV fusion antigen, E5, on the spore surface using CotB as the anchoring protein. E5 was designed by tandemly linking selected GP3, GP4, and GP5 fragments. The recombinant spores were evaluated in mice and piglets as part of a proof-of-concept immunogenicity study of an oral antigen-display platform, rather than a vaccine efficacy study. In mice, oral administration of RG induced antigen-specific serum IgG and intestinal secretory IgA responses, accompanied by detectable in vitro neutralizing activity against PRRSV R98 and NADC30-like strains. RG administration was also associated with changes in selected ileal cytokines and splenic CD3⁺, CD4⁺, and CD8⁺ staining patterns, indicating immune-related modulation following oral delivery. In piglets, RG similarly induced PRRSV E5-specific serum IgG and fecal secretory IgA responses and elicited detectable serum neutralizing activity against both R98 and NADC30-like strains. Overall, these findings support the preliminary immunogenicity and technical feasibility of engineered B. subtilis spores as a spore-based oral antigen-display platform for inducing systemic and mucosal antibody responses with detectable in vitro neutralizing activity. However, neither homologous nor heterologous PRRSV challenge experiments were performed; therefore, the present findings support immunogenicity but do not establish protection against infection or vaccine efficacy. This study provides a foundation for the further development of spore-based functional microbial systems for PRRSV-related mucosal antigen delivery and presentation.