Abstract

Introduction: Surface display of proteins on Bacillus subtilis has emerged as a promising strategy in vaccinology, leveraging its safety, gastrointestinal resilience, and capacity for efficient antigen presentation. Targeting Staphylococcus aureus, a pathogen reliant on alpha-toxin (Hla) for virulence, this study focuses on a detoxified variant, Hla_H35LH48L, to potentially neutralize toxicity while preserving immunogenicity. We investigated B. subtilis as an oral vaccine vector to display Hla_H35LH48L and elicit mucosal and systemic immune responses in mice.

Methods: The hla_H35LH48L gene was fused to the yhcR anchoring motif and integrated into the amyE locus of B. subtilis HT800F via double-crossover recombination, generating strain BsHT2315. Successful chromosomal integration was confirmed by PCR. Surface display of Hla_H35LH48L was verified through Western blot and bacterial-enzyme-linked immunosorbent assay (bactELISA). Swiss mice were orally administered BsHT2315, wild-type B. subtilis, or PBS (control). Serum IgG and intestinal IgA levels were quantified by ELISA.

Results: Western blot and bactELISA confirmed robust surface expression of Hla_H35LH48L on BsHT2315. Oral immunization with BsHT2315 induced a significant two-fold increase in intestinal IgA compared to controls (p < 0.05), indicative of mucosal immunity. Serum IgG levels also showed a modest but significant elevation (1.5-fold, p < 0.01), suggesting systemic response activation.

Conclusion: This study demonstrated the successful development of B. subtilis BsHT2315 as an oral vaccine vehicle for Hla_H35LH48L delivery. The strain triggered potent mucosal and systemic antibody responses, underscoring B. subtilis’s potential for cost-effective, needle-free vaccine platforms. Future work will explore protective efficacy against Staphylococcus aureus infection and scalability for clinical translation.