Japan The Schistosome Multi-epitope Vaccine Construct Exploration And Tsp-2 Antigen Identification

Schistosomiasis is one of the most serious parasitic diseases that infect approximately 200 million people living in the endemic areas of 76 countries and territories. Schistosomiasis japonica which is the one most hard to be prevented is still endemic in China today. Chemotherapy, using praziquantel (PZQ) play an important role in controlling the disease, but repeated infection and potential drug resistance render all chemotherapy-based control strategies inadequate. Therefore, safe and effective vaccine, alone or in combination with anthelmintic drugs would provide a major boost in the disease control program. However, a handful of antigens tested so far are not satisfied because of safety or efficacy which makes the identification of protective antigens and development of novel vaccines an urgent task.Using methods of bioinformatics, 18 B and/or T epitopes were selected from 8 key antigens of Schistosoma japonicum. Among them, 5 were identified in S. japonicum and another 13 were the homologous sequence of protective epitopes identified in Schistosoma mansoni. Five libraries with different polyepitope gene length were successfully constructed by the epitope shuffling technique. Each polyepitope library was shown the higher diversity of polyepitope genes based on the PCR analysis.With the polyepitope library immunization in mice, we have proved that the length of polyepitope gene affects the antigenicity of DNA library. Protective efficacy elicited by the five libraries were not so satisfied, probably because of the most epitopes selected were not truly identified in S. japonicum. Library L2 induced a moderate anti-fecundity effect in mouse model suggesting that only the library with appropriate polyepitope gene length can elicit protective immunity.According to the work above, we proved that the novel epitope shuffling technique can be widely used in constructing polyepitope library against various pathogens and confirmed the gene length of library which may induce protective immune response. Further, in order to construct polyepitope library against S. japonicum with high protection, vigorous endeavor should be made for protective epitope identification.Members of tetraspanin family expressed on the tegument of Schistosoma mansoni have been regarded as potential protective antigens, especial the novel tegumental antigen tetraspanin-2 (TSP-2) which induced as high as～60% protection in S. mansoni. So, the identification and evaluation of its homolog in S. japonicum were also carried out. Surprisingly, seven Sj-tsp-2 subclasses were identified according to the sequence variation in C and D variable regions, and hybrid Sj-tsp-2 cDNAs among different subclasses were also detected. 3D prediction revealed that the variable region was exposed on the surface of Sj-TSP-2 protein, suggesting this molecule were under positive selection, thus implicated that polymorphism of its potential ligands. Single worm RT-PCR showed that the transcription profiles of Sj-tsp-2 subclasses were highly variable in individual adult worms. All results above prompted that the Sj-tsp-2 was involved in immune evasion of S. japonicum.A semi-quantitative RT-PCR analysis revealed that Sj-tsp-2 gene was transcribed in cercariae, schistosomula, adult worms and eggs; however, Western blot analysis indicated that the Sj-TSP-2 protein was not expressed in eggs. In immunofluorescence assays (IFA), Sj-TSP-2 could be detected on the tegument of schistosomula, but not directly exposed to the host. However, IFA on live adult worms and cryosections showed that Sj-TSP-2 was obviously exposed on the surface of both male and female worms.Mice immunized with the recombinant protein of a single Sj-TSP-2 subclass showed no protection, while immunized with a mixture of seven recombinant Sj-TSP-2 subclasses provided a moderate protection. Those data implicated that the tegument protein Sj-TSP-2 may be involved in immune evasion and highly polymorphic of this molecule must affect its potential as a vaccine candidate.Thus, an impressive divergence of tegumental TSP-2 molecule was shown between the two species of S. mansoni and S. japonicum in above work. These results demonstrated that the homolog in S. japonicum of the protective antigen identified in S. mansoni may obtain no protection at all, thus indicated that living under the different environmental pressure, S. japonicum may adopt immune evasion strategies differed from that of S. mansoni. In some degree, this fact gives a circumstantial evidence to the result of the frontal part, underlining the importance of identification of specific protective antigens and epitopes for the development of S. japonicum vaccine.