Screening And Identification Of Th Cell Epitopes From Schistosomula Antigen Of Schistosoma Japonicum

Schistosomiasis is one of the most affected zoonotic parasitic diseases in our country, seriously affecting human health and socio-economic development. Hence, the control and prevention of the spread of schistosomiasis is significant. The traditional method of treatment of schistosomiasis is praziquantel chemotherapy drugs, combined with the control of infection sources as well as the strategy of environmental improvement and health education. But this treatment is costly, and requires a large number of professional and technical staff, and there can not prevent re-infection and drug resistance issues. Therefore, it is urgent to develop schistosome vaccine to replace the traditional control methods.But how efficiently and rationally screening and identified a protective antigen against Schistosoma japonicum has been a emergency need to overcome this bottleneck. Many studies indicate that radiation attenuated cercariae or schistosomula immunity in a variety of animal models have received a higher protective effect; Another study using the schistosomula that are from the irradiated cercariae and cultured in vivo to the lung stage and injected into the mice within the dermis, also lead to a high level of protective immune response, suggesting that some of these specific protein antigen from schistosomula can be seen as an important protective immune response inducer. In addition, some molecules containing the signal peptide sequence may be secreted into the extracellular, and thus there may be direct contact with the host immune systemagainst Schistosoma japonicum to induce immune responses and they may also be potential candidates for protective antigens. Therefore, in order to find out the protective antigen against Schistosoma japonicum candidate molecules efficiently and reasonablely, it imay be worth exploring to screening and identificate enrichment of irradiated schistosomula sequences, lung-stage schistosomula sequences, and sequences containing signal peptides.In this study, we use reverse vaccinology strategy, through the published literature and the Genome Center South to find our target sequences. They are as follows: high expression sequences of lung-stage schistosomula and 4 days irradiated enrichment sequences of Mannesmann Schistosoma. Through the method of BLAST we found the highest homology sequences of Schistosoma japonicum with those Mannesmann Schistosoma. Through the online server to identify some of these sequences contain a signal peptide. Organize these different characteristics sequences of Schistosoma japonicum to get 21 sequences which is both highly expressed in lung-stage schistosomula and containing the signal peptide; to get 2 sequences which is both highly expressed after fadiation and high expression in lung-stage schistosomula; to get 3 sequences which is both highly expressed after fadiation and high expression in lung-stage schistosomula. This providing a foundation for further Th cell epitope prediction snalysis.In order to acreening out candidate molecules that may be protective Th cell epitopes, immune informatics technology is used in the study to predict the candidate 26 sequences Th cell epitope. Use HLAPRED and MHCPred Sever based on the Matrix and the structure algorithm respectively to predict Th cell epitope which can connected with the HLAⅡalleles molecules .Use SYFPEITHI sever to predict 15 Th epitope peptide connected with the allele HLAⅡmolecules. Use MHCPred server to predict epitope peptide connected with HLAⅡmolecular of murine(H2d). Use the receptor binding ligand docking simulation server space based on the molecular dynamics method, to analog the binding situation of peptides and MHCⅡmolecules to get final candidate Th cell epitopes: NH-NNSVVIEKSHFLNVL-COOH[AAW26577]; NH-MFGYDKVLPMNSGVE-COOH[AAW26182]; NH-ISVFLFVLTFQYIIS-COOH[AAW24929]; NH-SFFFFLIIHNKIGLA-COOH[AAW25469]; NH-GRVWQVIILTGSYWM-COOH[EZ000175]; NH-MEAFKTFDREGQGFI-COOH[AAW26398]This providing the theoretical foundation for experimental identification work.To confirm the sequence analysis and experimental epitope prediction results, the peptides were synthesized using the multiple antigenic peptide (MAP) approach. The multiple copies of a 15 amino acid peptide synthesized by solid-phase Fmoc chemistry are grafted to an 4-branched polylysine core peptide (PCP). Use RT-PCR method to detect the expression condition of candidate epitope peptides in the 3-days lung-stageschistosomula at mRNA levels;Flow cytometry is to detect if four of the six peptides can binding to antigen presenting cells (APCs) effectively. The modified MTT method (CCK-8) is to test if the candidate epitopes can stimulate the immunelyphocyte proliferation in vitro; Intracellular cytokine–producing CD4+ cells were measured by flow cytometry (FCM).Through quantitative real-time PCR analysis, we found that all of the six source sequences of predicted peptides can express in normal day 3 schistosomula from BALB/c mice infected with S. Japonicum. We measured the direct binding of the four peptides (i.e. P1, P2, P5 and P6 ) to APCs from BALB/c mouse strain using a fluorometric method, the result shows about 43%-88% of the cells were labeled with above four biotinylated peptides.Each unmodified peptide inhibited the binding of its biotinylated peptide with the inhibition ratio of 56%-93.66%; The inhibition by anti-I-Ad and anti-I-Ed was 68%-87% and 49%-75% respectively, which is similar and higher than that of the positive peptide, suggesting that the four peptides can bind to both I-Ad and I-Ed molecules on APC cells of BLAB/c mice. The results of lymphocyte proliferation experiments showed the stimulation index of five predicted peptide(except P3) was significantly higher than adjevant control group; and the stimulation index of P2, P4 and P6 peptide groups, were significantly higher than that of poly-lysine backbone group(PCP control group), indicating that they can stimulate the immune mice with different degrees of lymphocyte proliferation. the proportions of IFN-γ-producing CD4+ cells were higher than those of IL-4-producing cells in splenocyte cultures restimulated with P1, P2 and P5 peptides. However, the proportions of IL-4-producing CD4+T cells were higher than those of IFN-γ-producing splenocytes when the cells were restimulated with the P4 peptides; and P3 and P6 peptides were not able to induce peptide-specific immune mice have CD4 + T cell-related cytokines. Therefore, it is concluded that the P1, P2 and P5 candidate peptides are likely to be Th1-type epitopes, while P4 peptide may be of Th2 type.Overall, through analysising Schistosoma japonicum transcripts omics data as well as immune informatics prediction work, we screened out 26 japonicum schistosomula molecules and six Th cell epitopes from these molecules. And we identified the immunogenicity of predicted candidate epitopes through the Molecular and Cellular Immunology which lay a foundation for the rapid and efficient screening of Schistosoma japonicum protective T cell antigen or epitope.