IDENTIFICATION OF NATURALLY PROCESSED SMANSONI PEPTIDES

血吸虫病，一种由曼氏血吸虫引起的衰弱疾病， 据估计，全球有2-3亿人受到影响。临床和 实验证据支持这样一种观点，即自然免疫 在受感染的主机中存在，这表明应该有可能 开发有效的血吸虫病疫苗。关于免疫的研究 在感染期间或接种疫苗后使用 在小鼠和大鼠模型中减毒的寄生虫表明，CD4+T- 淋巴细胞在保护性免疫中起主要作用。外源蛋白质 分子被巨噬细胞等抗原提呈细胞摄取。 和B淋巴细胞，并在这些细胞内通过去折叠和 蛋白质分解为短肽片段。然后这些碎片就会 显示在细胞表面，与MHC II类分子结合。装订 在MHC分子的背景下这些多肽的T细胞受体 触发T辅助(Th)细胞免疫反应的级联反应。雅致 方法论的发展使我们有可能摆脱这些 酸提取纯化MHC分子中的多肽及其分析 通过微测序。这项提议的目标是孤立和 用这个识别血吸虫起源的重要T细胞表位 新的方法方法。这项建议的具体目标是： 1)MHC-II类结合曼氏杆菌的分离与鉴定 多肽。洗脱的多肽将被测序并大量 我们将合成合适的多肽，以供进一步研究。2)至 使用合成肽在小鼠体内诱导免疫反应。合成的 在阳性增殖反应基础上构建的多肽 和Th1型细胞因子分泌在T细胞筛选检测序列中 与曼氏沙门氏菌抗原的同源性，将用于解决这一目的 建议和3)研究Balb/c的体内保护性反应， 人工合成蛋白免疫C57BL/6、SJL和CBA/J小鼠 多肽。我们之前给出的数据显示，多肽被洗脱 从NP40提取物冲击的A20细胞系MHC II类分子中提取 血吸虫刺激肿大淋巴结的增殖性反应 从免疫BALB/c和C57BL/6小鼠中获取细胞。在.期间 在1997年10月至1998年6月的试点资金期间，我们有 从II类分子中成功分离洗脱出的多肽部分 采用反相高效液相色谱法。此外，我们还测试了 刺激抗原特异性T细胞增殖的单组分 和细胞因子分泌反应。已经分析了单独的分数 由我们的合作者约翰·科利根博士、美国国立卫生研究院和美国国家卫生研究院正在进行测序。 弗吉尼亚大学的唐纳德·亨特作为一名 合作者，使用微毛细管高效液相色谱-电喷雾电离 三重四极杆串联质谱仪。因此，我们有信心 我们提交的这个项目是可行的，它可以 顺利完成。

Schistosomiasis, a debilitating disease caused by Schistosoma mansoni, is estimated to affect 200-300 million people worldwide. Clinical and experimental evidence supports the notion that natural immunity does exist in the infected host, indicating that it should be possible to develop an effective vaccine against schistosomiasis. Studies on immune response modulation during infection or following vaccination using attenuated parasites in mice and rat models indicate that CD4+ T- lymphocytes play a major role in protective immunity. Foreign protein molecules are taken up by antigen-presenting cells such as macrophages and B-lymphocytes and are processed within these cells by unfolding and proteolysis to short peptide fragments. These fragments are then displayed on the cell surface, bound to MHC class II molecules. Binding of T-cell receptor to these peptides in the context of MHC molecules triggers a cascade of T-helper (Th) cell immune responses. Elegant methodological developments have made it possible to elute these peptides from purified MHC molecules by acid extraction and to analyze them by microsequencing. The goal of this proposal is to isolate and identify important T-cell epitopes of schistosome origin using this novel methodological approach. The Specific Aims of this proposal are: 1) Isolation and identification of MHC class II bound S. mansoni peptides. Eluted peptides will be sequenced and large quantities of appropriate peptides will be synthesized for further studies. 2) To elicit immune response in mice using synthetic peptides. Synthetic peptides, constructed on the basis of positive proliferation response and Th1-type cytokine secretions in the T-cell screening assay sequence homology with S. mansoni antigen, will be used to address this aim of the proposal and 3) To study in vivo protective response in Balb/c, C57BL/6, SJL and CBA/J mice following immunization with synthetic peptides. We had previously presented data showing that peptides eluted from MHC class II molecules of A20 cell line pulsed with NP40 extract of schistosomule stimulate a proliferative response in bulk lymph node cells harvested from vaccinated BALB/c as well as C57BL/6 mice. During the period October 1997- June 1998 of pilot funding, we have successfully separated peptide fractions eluted from class II molecules by reverse phase HPLC. Further, we have tested the ability of individual fractions to stimulate antigen-specific T cell proliferation and cytokine secretion response. Individual fractions have been analysed by our collaborator Dr. John Coligan, NIH and are being sequenced by Dr. Donald Hunt, University of Virginia who has joined this project as a collaborator, using microcapillary HPLC-electrospray ionization with triple quadrupole-tandem mass spectrometry. We are therefore confident that this project we have submitted is a feasible one and that it can be completed successfully.