Systemic and local mucosal immune responses induced by orally delivered Bacillus subtilis spore expressing leucine aminopeptidase 2 of Clonorchis sinensis.

Human clonorchiasis caused by Clonorchis sinensis (C. sinensis) has been increasingly prevalent in recent years so that an effective measure is essential and urgent to control the infectious disease. Oral delivery of antigens from C. sinensis may be an important approach to effectively induce both systemic and local immune responses to anti-infection of the parasite. In the current study, we used Bacillus subtilis (B. subtilis) spores as a delivery vehicle to introduce leucine aminopeptidase 2 of C. sinensis (CsLAP2), an excretory/secretory antigen with high immunogenicity, expressing on their surface. SDS-PAGE, western blotting, and flow cytometry indicated that CsLAP2 was successfully expressed on the surface of B. subtilis spores (CotC-CsLAP2 spores). BALB/c mice were treated with spores intragastrically. On day 31 after the treatment, we found that mice intragastrically treated with CotC-CsLAP2 spores exhibited higher IgG, IgG1, IgG2a, and IgA level in sera as well as higher sIgA level in bile and intestinal lavage fluid compared to mice orally administrated with spores not expressing CsLAP2 (CotC spores) and naïve mice. The peak titer of IgG/IgA presented on day 31/49 after oral administration. IgG1 level was lower than IgG2a in group administrated with CotC-CsLAP2 spores. sIgA-secreting cells were obviously observed in intestinal epithelium of mice orally treated with CotC-CsLAP2 spores. After incubated with CotC-CsLAP2, the levels of IFN-γ, IL-6, IL-10, IL-17A, and TNF significantly increased in the supernatant of splenocytes isolated from mice orally treated with CotC-CsLAP2 spores, while there was no statistically significant difference of IL-4 level representing Th2 response among the groups. Our study demonstrated that oral administration of CsLAP2 delivered by B. subtilis spore elicited obvious systemic and local mucosal immunity. Secretory IgA and Th1-Th17 cellular immunity might involved in mechanisms of the immune response.