Bacillus subtilis spore with surface display of paramyosin from Clonorchis sinensis potentializes a promising oral vaccine candidate

Background: Clonorchiasis caused by Clonorchis sinensis has become increasingly prevalent in recent years. Effective prevention strategies are urgently needed to control this food-borne infectious disease. Previous studies indicated that paramyosin of C. sinensis (CsPmy) is a potential vaccine candidate. Methods: We constructed a recombinant plasmid of PEB03-CotC-CsPmy, transformed it into Bacillus subtilis WB600 strain (B.s CotC CsPmy), and confirmed CsPmy expression on the spore surface by SDS-PAGE, Western blotting and immunofluorescence assay. The immune response and protective efficacy of the recombinant spore were investigated in BALB/c mice after intragastrical or intraperitoneal immunization. Additionally, biochemical enzyme activities in sera, the intestinal histopathology and gut microflora of spore-treated mice were investigated. Results: CsPmy was successfully expressed on the spore surface and the fusion protein on the spore surface with thermostability. Specific IgG in sera and intestinal mucus were increased after intraperitoneal and intragastrical immunization. The sIgA level in intestinal mucus, feces and bile of B.s-CotC-CsPmy orally treated mice were also significantly raised. Furthermore, numerous IgA-secreting cells were detected in intestinal mucosa of intragastrically immunized mice. No inflammatory injury was observed in the intestinal tissues and there was no significant difference in levels of enzyme-indicated liver function among the groups. Additionally, the diversity and abundance of gut microbiota were not changed after oral immunization. Intragastric and intraperitoneal immunization of B.s-CotC-CsPmy spores in mice resulted in egg reduction rates of 48.3 and 51.2% after challenge infection, respectively. Liver fibrosis degree in B.s-CotC-CsPmy spores treated groups was also significantly reduced. Conclusions: CsPmy expressed on the spore surface maintained its immunogenicity. Both intragastrical and intraperitoneal immunization with B.s-CotC-CsPmy spores induced systemic and local mucosal immune response in mice. Although both intragastric and intraperitoneal immunization elicited a similar protective effect, intragastric immunization induced stronger mucosal immune response without side effects to the liver, intestine and gut microbiota, compared with intraperitoneal immunization. Oral immunization with B. subtilis spore expressing CsPmy on the surface was a promising, safe and needle-free vaccination strategy against clonorchiasis.