Recombinant Bacillus subtilis spores expressing cholera toxin B and ovalbumin prevent ovalbumin-specific food allergy in mice by upregulating regulatory T cells and modulating gut microbiome flora.

BACKGROUND: Although oral immunotherapy has shown clinical efficacy in treating food allergies, its broader implementation is constrained by the occurrence of adverse effects. Consequently, inducing allergen-specific immune tolerance during early life can be a preventive strategy to reduce the development of food allergy. OBJECTIVE: Here, we developed a novel fusion protein cholera toxin B (CTB)-ovalbumin (OVA) expressed on Bacillus subtilis (B.s-CotC-CTB-OVA) spore surface and investigated whether B.s-CotC-CTB-OVA spores prevent OVA-induced food allergy in a mouse model and explored the potential underlying mechanisms. METHOD: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to confirm that CTB-OVA was expressed on B. subtilis spores. Female BALB/c mice were orally administered with B.s-CotC-CTB-OVA spores and B. subtilis spore control (B.s-CotC and B.s-CotC-CTB) for 4 weeks. Then, sensitization and challenge with OVA were performed on mice. Fecal OVA-secretory IgA (sIgA) and serum OVA-IgE, IgG1, and IgG2a levels were measured by enzyme-linked immunosorbent assay (ELISA). The gut microbiome was analyzed by 16S rDNA sequencing. After challenge, diarrhea score, anaphylactic reactions score, splenocyte interleukin (IL)-10, IL-4, and interferon-γ (IFN-γ), and Treg levels were measured. mRNA of IL-10, IL-4, IFN-γ, and Foxp3 were measured. Fecal microbiota transplant (FMT) was used to explore the mechanisms of microbiome in B. subtilis on food allergy. RESULTS: Recombinant CTB-OVA was successfully expressed on the surface of B. subtilis. Oral administration of B.s-CotC-CTB-OVA can increase fecal OVA-sIgA, alleviate food allergy symptoms, and decrease serum OVA-IgE in mice with significance (p < 0.05). Moreover, oral administration of B.s-CotC-CTB-OVA can significantly reduce serum OVA-IgG1, OVA-IgG2, IL-4, spleen mast cells, and eosinophil levels and significantly increase serum IL-10 and Treg levels (p < 0.05). Additionally, microbiome analysis shows that oral administration of B.s-CotC-CTB-OVA can significantly increase the relative abundance of Muribaculaceae and significantly decrease the relative abundance of Alistipes. FMT partially reproduced the reduction in serum OVA-specific IgE, but did not significantly improve allergic symptom or diarrhea scores, suggesting that gut microbiota alterations may partially contribute to the immunological effects of B.s-CotC-CTB-OVA. CONCLUSION: These findings suggest that B.s-CotC-CTB-OVA spores may serve as a preventive oral antigen-delivery strategy to promote antigen-specific immune regulation and partially modulate microbiota-associated immune responses in OVA-induced food allergy.