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

Abstract

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.

Clonorchis sinensis Co-infection Could Affect the Disease State and Treatment Response of HBV Patients

“…It was suggested that ESPs are immunogenic, stimulate inflammation and promote proliferation, and suppress apoptosis [ 32 ]. Most proteins belonging to ESPs, including Cs RNASET2, Cs LAP2, and Cs NOSIP, contributed in eliciting Th2 immune response in mice [ 36 – 38 ]. Cytokines are important mediators in the regulation of the immune response.…”

The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine protease

“…The DNA sequence was inserted into the multiple clone site of the pBluescript II SK (−) plasmid [15, 27] after digestion with Sal I and Hind III. The coding sequence of Cs CP was amplified from the previously constructed pET28a (+)- Cs CP plasmid using specific primers (F: 5′-CCC AAG CTT AGC AAC ATT CCT GAA TCA G-3′, R: 5′- TAC GAG CTC TCA CAA GAT GAT CGA GGT G-3′) containing restriction sites for Hind III and Sac I (underlined).…”

“…The levels of IgG, IgG1 and IgG2a in serum were measured by an ELISA assay as described previously [15, 27]. Plates were coated with 5 μg/ml purified r Cs CP (0.05 M carbonate-bicarbonate buffer, pH 9.6) at 100 μl per well and incubated at 4 °C overnight.…”

Immunobiology of parasitic worm extracellular vesicles

“…As type 2 immune responses are implicated with enhancing innate and acquired immunity to helminths which mediates parasite expulsion, future work on effective anti‐helminth vaccines should focus on the induction of a robust type 2 response using suitable adjuvants and mucosal delivery strategies that present EV proteins in a manner that replicates their presentation in an infectious setting. For example, mucosal delivery strategies with orally administered nanoparticles or attenuated bacteria (or their spores) bearing surface exposed recombinant proteins derived from EVs warrants more in‐depth investigation. In the long term, package of vaccine candidates into synthetic EVs could serve as an ideal delivery system and adjuvant and would permit protection of antigens from digestive proteases in the alimentary canal while improving antigenicity by targeting specific types of antigen‐presenting cells …”