Molecular characterization and identification of Th1 epitopes of a Schistosoma japonicum protein similar to prosaposin.

The tegument of schistosomula contains T cell antigens that might simulate the protective mechanisms of the radiation-attenuated vaccine in a mouse model of schistosomiasis. Immune mechanisms mediated by the CD4+ Th1 response are important in the RAV model. To rapidly identify Th1 epitopes in molecules from the Schistosoma japonicum schistosomula tegument, this study analyzed S. japonicum proteomics data. Preliminary experiments identified a protein similar to prosaposin (SjPSAP) from the tegument of schistosomula. We confirmed that SjPSAP was present in the tegument of the parasite using an indirect immunofluorescence assay. We then identified Th cell epitopes in SjPSAP using in silico prediction combined with experimental validation. From the SjPSAP sequence, we used several algorithms to predict 11 promiscuous Th cell epitopes that might bind to both murine and human MHC class II molecules. To validate the in silico predictions, proliferation and cytokine production profiles of spleen lymphocytes from BALB/c mice immunized with the 11 predicted peptides were measured in vitro using a modified methyl thiazolyl tetrazolium assay and flow cytometry. The results showed that 4 of the 11 predicted peptides induced a recall CD4+ Th1 response in vitro. We measured direct binding of the four peptides predicted to induce a response to antigen-presenting cells from BALB/c mice using a fluorometric method and found that the peptides bound to both I-Ad and I-Ed mouse molecules. These results demonstrated that potentially protective Th1-type epitopes in SjPSAP molecules could be identified rapidly by combining in silico prediction with experimental validation. This strategy could be a fast method for identifying Th1 epitopes in a schistosoma antigen with features such as large size or poor expression of recombinant antigens.

Identification of Th1 epitopes within molecules from the lung-stage schistosomulum of Schistosoma japonicum by combining prediction analysis of the transcriptome with experimental validation.

The lung-stage schistosomulum has been regarded as the main target of protective immunity induced by radiation-attenuated vaccines (RAV) in the mouse model of schistosomiasis, and immune mechanisms mediated by the CD4+ Th1 response play a major role in the RAV model. To identify Th1 epitopes rapidly within molecules from the lung schistosomulum of Schistosoma japonicum, in the present study we analyzed transcriptome data from normal and radiation-attenuated lung schistosomula of S. japonicum and Schistosoma mansoni. We selected six genes with high levels of expression of their transcripts as sample sequences from the lung schistosomula. From these six sequences, by using different algorithms, we predicted six promiscuous Th cell epitopes that are capable of binding to both murine and human MHC class II molecules. To validate our in silico prediction experimentally, first, the gene expressions of the six sequences in day 3 lung-stage schistosomula were assessed using reverse-transcription PCR (polymerase chain reaction) analysis. The result showed that all six sequences predicted can be expressed in normal day 3 schistosomula. Second, we measured the direct binding of the four peptides predicted above to APCs (Antigen Presenting Cells) from the BALB/c mouse strain using a fluorometric method, and found that the four peptides could bind to both I-Ad and I-Ed molecules of the mice. Finally, the proliferation and profiles of cytokine production by spleen lymphocytes from the BALB/c mice immunized with the six predicted peptides were detected in vitro using modified MTT (Methyl Thiazolyl Tetrazolium), and flow cytometry methods, respectively. The results showed that three of the six predicted peptides could induce a recall CD4+ Th1 response in vitro. These results demonstrate that potential Th1-type epitopes can be identified rapidly by a combination of in silico analysis of transcriptomes of lung-stage schistosomula with experimental validation.

In silico prediction of binding of promiscuous peptides to multiple MHC class-II molecules identifies the Th1 cell epitopes from secreted and transmembrane proteins of Schistosoma japonicum in BALB/c mice.

It has not so far been possible to identify rapidly and effectively the anti-schistosomiasis Th cell epitopes that are capable of simulating IFN-γ (Interferon-gamma)-mediated Th1-type protective immunity in response to radiation-attenuated schistosome cercaria. With the advance of the omics studies of schistosomes, an approach that used reverse vaccinology probably resolved the above problems. In this "proof-of-principle" study, first, we selected 31 secreted or transmembrane protein sequences sampled from sequences of the transcriptome of Schistosoma japonicum, and analyzed characteristics of these proteins by using conventional bioinformatics tools. Second, putative promiscuous Th cell epitopes within these proteins were predicted using three to four different immuno-informatics algorithms for the prediction of MHC (Major Histocompatibility Complex) class-II binding peptides. We predicted using these in silico approaches promiscuous Th cell epitopes that are capable of binding to both murine and human MHC class-II molecules. To validate our in silico prediction experimentally, BALB/c mice were immunized with the five predicted peptides, and the proliferative responses and cytokine production of lymphocytes from the immunized BALB/c mice were assessed in vitro by modified MTT (Methyl Thiazolyl Tetrazolium), ELISA (Enzyme-linked Immunosorbent Assay) and flow cytometry methods. The results showed that two of the five predicted peptides could induce a Th1-type response in vitro. These results suggest that promiscuous Th1 cell epitopes from secreted or transmembrane proteins of S. japonicum can be identified using a strategy of reverse vaccinology.