Data Sheet 1_Antigen-displaying probiotic Bacillus subtilis spores induce subtle, strain-dependent immunomodulation in fish.docx

Despite the availability of commercial vaccines against several pathogens, infectious diseases continue to cause substantial economic losses in aquaculture. Current vaccine development focuses on exploring antigen delivery systems that enable efficient needle-free, mass vaccination. Bacterial spores offer a promising platform for oral vaccine delivery, as they are highly resistant structures that can act as adjuvants and antigen carriers. This technology has been explored in recent years, mostly using spores from laboratory strains, for which a variety of genetic tools have been optimized. The use of spores of autochthonous probiotic strains for displaying antigens remains to be further explored. In this study, we engineered two fish-gut Bacillus subtilis strains (FI314 and FI442) with probiotic potential to display the immunogenic proteins OmpK or TolC of Vibrio spp. Their immunomodulatory effect was evaluated using in vitro, ex vivo, and in vivo approaches. In RTgutGC cells, both FI314 and FI314-TolC spores induced an up-regulation of innate immune markers, including il1b and il8, while FI314 spores down-regulated casp3a2 expression. These effects were not observed with the probiotic FI442. In European seabass gut explants, FI314-TolC spores induced the expression of il10, while all spores induced the upregulation of ifng after bacterial challenge with V. anguillarum. In vivo, however, feeding European seabass with diets containing FI314, FI314-OmpK, or FI314-TolC spores for 30 days did not elicit a robust adaptive immune response, as indicated by the lack of significant modulation of immune-related genes and unchanged serum IgM levels. RNA-seq analysis of the distal intestine showed that FI314 spores induced a down-regulation of cell proliferation pathways. while OmpK-carrying spores affected innate immunity pathways. The results of this study indicate that the immunomodulatory effects of autochthonous probiotics are strain dependent. FI314 antigen-displaying spores were insufficient to induce an effective adaptive immunity, under the conditions tested. This study emphasizes the importance of optimizing bacterial strain selection, antigen choice, and immunization regimen when designing oral spore-based vaccines for fish.