日本血吸虫多表位疫苗构建探索和TSP-2抗原鉴定

【作者】 蔡鹏飞；

【导师】 王恒；

【作者基本信息】 中国协和医科大学 ， 病原生物学， 2008， 博士

【摘要】 血吸虫病是世界上严重危害人类健康的寄生虫病之一,流行于世界上76个国家和地区,目前有2亿感染者。流行于我国的日本血吸虫病是所有血吸虫病中防治难度最大的一种。利用吡喹酮治疗在控制该病的过程中起到重要作用,但治疗后的反复感染,及反复化疗可能产生抗药性问题,使得仅靠单一药物治疗无法从根本上控制血吸虫病的传播。研发安全有效的抗血吸虫疫苗,单独使用或结合化疗将极大地推动血吸虫病的防治工作。但早期研制的疫苗,从安全性或保护效果来看并不令人满意。因此,新型抗原的鉴定和新型疫苗的研制是一项十分紧迫的任务。本研究通过生物信息学方法,从日本血吸虫的8个关键抗原中筛查18个表位,其中5个在日本血吸虫中鉴定,其余13个为曼氏血吸虫中鉴定的表位在日本血吸虫中的同源序列。利用表位改组技术成功构建5个不同长度的随机串联多表位人工抗原文库,并证明文库具有良好的表位串联多态性。不同文库免疫小鼠后抗体水平的检测,表明多表位基因的长度对文库的抗原性具有显著的影响。保护性实验结果显示5个文库的保护效果并不理想,可能与所选的多数表位并未在日本血吸虫中得到有效证实有关。文库L2取得部分的抗生殖作用,表明只有合适长度的多表位基因文库才有可能诱导较好的免疫保护作用。通过上述工作,证实新型表位改组技术能应用于不同病原体多表位文库的构建,确定可能诱导保护性免疫反应的多表位基因长度;同时提示要构建具有高保护性的日本血吸虫多表位人工抗原文库,仍有待于保护性表位的鉴定。曼氏血吸虫表膜四次跨膜蛋白家族成员被认为是具有保护潜力的抗原分子,其中是曼氏血吸虫新型抗原TSP-2更是获得高达～60%的保护力,本研究对Sm-TSP-2在日本血吸虫中的同源分子展开鉴定和评估。研究证实日本血吸虫Sj-TSP-2分子存在广泛变异,根据C、D变异区不同,可分为七个亚类,同时存在不同亚类之间重组的杂合分子;三级结果预测显示主要变异区暴露于分子的表面,表明此分子受正选择作用,并提示其配体的多样性;单一成虫RT-PCR显示Sj-tsp-2亚类表达谱在个体成虫中存在极大的差异,以上研究结果提示Sj-stp-2可能参与日本血吸虫的免疫逃避。半定量RT-PCR表明Sj-tsp-2基因在日本血吸虫尾蚴、童虫、雌雄成虫和虫卵中都有不同水平的转录;但Western blot分析显示Sj-TSP-2蛋白并不在虫卵期表达。免疫荧光定位实验表明Sj-TSP-2定位于肺期童虫的表膜,但在天然状态下并不暴露;而活体成虫及冰冻切片免疫荧光实验则证实Sj-TSP-2分子暴露于雌雄成虫的体表。免疫保护实验显示单一Sj-TSP-2亚类重组蛋白不能获得任何的保护效果,所有亚类重组蛋白的混合物也只获得较低的保护力,再次提示Sj-TSP-2分子与免疫逃避相关,变异如此广泛的Sj-TSP-2并不适合成为日本血吸虫的候选抗原。以上工作显示表膜蛋白TSP-2在曼氏血吸虫和日本血吸虫中极大的差异性,说明曼氏血吸虫的保护性抗原,其在日本血吸虫中的同源分子并不一定同样具有保护性;提示在不同环境压力下,日本血吸虫可能采取了不同于曼氏血吸虫的免疫逃避策略,在一定程度上佐证了前一部分的结果,进一步强调日本血吸虫特异的保护性抗原和表位的鉴定对于日本血吸虫疫苗的研制的重要性。更多还原

【Abstract】 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.更多还原