Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

Abstract

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj)]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Fig. 1A. western blotting of 81 KDa of chimeric protein by horseradish peroxidase-conjugated anti-glutathione-S-transferase protein (GST) [Lane M: prestained protein molecular weight marker (Vivantis, PR0602); GST-ChMEA: chimeric protein]; B: [Lane M: midrange protein molecular weight marker) Promega, USA); A: supernatant after purification; B: first washing by binding buffer; C: chimeric antigen purified by GST-beads)].

Fig. 2. production of interferon (IFN)-γ, interleukin (IL)-4 and IL-10 by splenocytes isolated from mice immunised with the chimeric protein, as tested by the sandwich ELISA kit (Quantikine R&D). A: level of IFN-γ; B: level of IL-4; C: level of IL-10; Adj: mice immunised with adjuvant; Cm [glutathione-S-transferase protein (GST)]: cytokine levels in the absence of GST; Cm (GST-ChMEA): cytokine levels in the absence of GST-ChMEA; GST: cytokine levels in the presence of GST in mice immunised with the GST protein; GST-ChMEA: cytokine levels in the presence of the GST-ChMEA in mice immunised with the GST-ChMEA protein. Box plot designed by SPSS 11.5. Asterisk means p < 0.001.

Fig. 3. immunisation of mice with glutathione-S-transferase protein (GST) induced a significant increase in spleen cell number (A) and in spleen size (B) as well as a slight increase in size of mesenteric lymph nodes (C). C-adjuvant (Adj): injected mice with adjuvant; control-phosphate-buffered saline (PBS): injected mice with phosphate buffer; GST: injected mice with GST protein alone; GST-ChMEA: injected mice with the chimeric protein; *: p < 0.001; **: p < 0.006.