Identification Of T Cell Epitopes In Antigens Of Schistosoma Japonicum (Chinese Mailand Strain)

Schistosomiasis, the second major parasitic disease in the world after malaria, affects 200 million people. Vaccine strategies represent an essential component of the control of this chronic debilitating disease. Despite the fact that progress has been relatively slow, the successful use in animals of attenuated vaccines combined with recent encouraging results using defined antigens derived from Schistosoma japonicum suggests that development of a safe and effective vaccine is feasible, and has been targeted as a priority by the World Health Organization.Cellular immune response generated after antigen stimulation of lymphocyte populations can be characterized by the distinct cytokines that are produced. Many vaccine studies conducted in mice and human support the type-1-cytokine-mediated mechanisms. The evolution of vaccine strategies has seen a move from whole organisms to recombinant proteins, and further towards the ultimate in minimalist vaccinology, the epitope. The T-cell epitopes of antigens in S.mansoni have been extensively studied, but identification of the epitopes in the antigens of S.japonicum remains inadequate.We previously investigated the molecules of mitochondria-related protein(Sj338) and 22.6 kDa antigen(Sj22.6) of S.japonicum, and found that they could partly protect mice from later challenge infection. The mimitopes of these two molecules protect mice better than those from one molecule.The identification of T cell epitopes, based on MHC-peptide-binding assay or overlapping peptide approach is costly and labor-intensive. We report here the use of two computer-based prediction algorithms, which are readily available in the public domain(internet), to identify T cell epitopes. In this study, we identified the T cell epitopes harbored in Sj338, Sj22.6, triose-phosphate isomerase(SjTPI) , 97 kDa paramyosin (Sj97) of Schistosoma japonicum (Chinese Mainland Strain) based on the methodsinvolving the in vitro isolation and restimulation of lymphocytes.The primary structure of these molecules was analysed using various predictive algorithms including TEPITOPE and SYFPEITHI softwares. The first predictive algorithm, "TEPITOPE", was used to predict promiscuous T cell epitopes of Sj338 and Sj22.6. The second algorithm, "SYFPEITHI" , was used to predict H-2k specific MHC class II binding peptides of Sj97. The amino acid sequence of Sj97 was analyzed on this computer programs for the existence of 15-amino acid peptides predicted to bind to H-2AK or H-2EK. The number of candidate peptides was narrowed down by establishing a cutoff value of algorithms scores. The analysis resulted in 14 candidate peptides for T cell epitopes.The identification of epitopes using synthetic peptide approach is costly, thus we attempted to use recombinant method. The oligonucleotides of candidate epitopes were designed, synthesized and inserted into the multiple cloning site of plasmid pET-32c(+). The recombinant plasmids were transformed into Escherichia coli BL21 and identified by endonuclease digestion and sequencing. The positive clones containing the recombinant plasmids could express specific fusion proteins (epitope-Trx, MW 20 kDa) as a soluble form after the isopropyl-beta-D-thiogalactoside induction. The fusion protein with 6 His could be coupled with NTA resin specifically, and purified by elusion of the column with buffer containing imidazole. Therefore, the fusion proteins were purified by nickel-affinity chromatography to homogeneity.The purified Trx fusion proteins(recombinant peptides) were incubated with the primed lymph node cells and splenocytes of C3H/HeJ or C57BL/6 mice, respectively. To assess proliferation, 2.5 106/ml single-cell suspensions were cultured in a volume of 200 l for a total of 72 hours in the presence of defined antigens. During the last 12 ~ 16 hours of culture, 0.5 Ci of tritiated thymidine was added to each well. The radioactivity of incorporation into DNA was measured by liquid scintillation spectoscopy after harvesting the cell cultures onto glass fiber filters. The SI(s...