Complement Regulation in Trypanosoma Cruzi

克鲁兹锥虫的补体调节

• 批准号: 6435114
• 负责人: Karen A Norris
• 金额: $ 26.69万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6435114
• 关键词: Trypanosoma cruzi; active immunization; binding proteins; biotechnology; complement; complement pathway; drug delivery systems; drug screening /evaluation; enzyme complex; enzyme inhibitors; glycoproteins; host organism interaction; immunoregulation; laboratory mouse; microorganism culture; microorganism immunology; molecular cloning; monoclonal antibody; mucosa; nonhuman therapy evaluation; oral administration; polymerase chain reaction; protozoal antigen; protozoal vaccine; recombinant proteins; trypanosomiasis; vaccine development; vector vaccine

DESCRIPTION: (provided by the applicant): The goal of this research is the molecular and immunologic characterization of the 160 kDa complement regulatory protein (CRP) of the protozoan parasite, Trypanosoma cruzi, the causative agent of Chagas disease. After infection of the vertebrate host, the bloodstream stage trypomastigote is capable of evading host alternative and classical complement activation pathways, allowing dissemination to targettissues and intracellular replication. This is accomplished through the production of adevelopmentally regulated surface glycoprotein, CRP that restricts complement activation atthe level of C3 convertase formation. Our hypothesis is that immunologic neutralization of theCRP contributes to destruction of the extracellular bloodstream stage, and thus will contribute to protection from disease. We have shown that immunization with plasmid DNA encoding the full length CRP structural gene elicits neutralizing antibodies and protects against a lethal challenge in a murine model of Chagas disease. Three specific aims are designed to optimize the design of vaccine candidates based on the CRP gene and to characterize the immune responses to the protein, in vivo. Specific Aim 1 will provide a molecular analysis of the active domains of CRP and the binding interaction between CRP and its ligand, the complement component C3b. The functional analysis will be carried out using mutant CRP constructs expressed in mammalian cells and assayed for complement restriction activity and C3b binding. In addition, we will use neutralizing anti-CRP monoclonal antibodies and CRP-binding peptides to provide a finer map of activity. Specific Aim 2 will focus on an evaluation of CRP-DNA based vaccines in a murine model of Chagas disease. In these studies we will compare three expression vectors, optimize immunization protocols, and evaluate the range of protection using multiple mouse and parasite strains. In addition, we will examine oral and mucosal vaccine delivery systems and mucosal, sub-lethal challenge regimens. The goal of Specific Aim 3 is to analyze the immune effector mechanisms involved in the pmtective anti-CRP response. The role of immune antibodies will be evaluated in DNA immunized mice to determine if conformationally dependent epitopes are essential to the induction of a protective response. Passive transfer and challenge studies will be conducted and we will test vaccinated mice for the presence of CRP-specific cytotoxic T cells using CRP-expressing target cells. The successful completion of these aims will provide the first functional and immunological evaluation of a critical virulence factor of T. cruzi and provide information and reagents for the use of CRP as a vaccine candidate.

描述：（由申请人提供）：本研究的目标是 160 kDa 补体调节的分子和免疫学特征 原生动物寄生虫克氏锥虫（病原体）的蛋白质 (CRP) 恰加斯病。脊椎动物宿主感染后，血流 阶段锥鞭毛体能够逃避宿主替代和经典 补体激活途径，允许传播到目标组织和 细胞内复制。这是通过生产来实现的 发育调节表面糖蛋白，限制补体的 CRP C3转化酶形成水平的激活。我们的假设是 CRP 的免疫中和有助于破坏 细胞外血流阶段，因此将有助于保护免受 疾病。我们已经证明，用编码完整序列的质粒 DNA 进行免疫 长度 CRP 结构基因引发中和抗体并防止 恰加斯病小鼠模型中的致命挑战。三个具体目标是 旨在优化基于CRP基因的候选疫苗设计， 表征体内对该蛋白质的免疫反应。具体目标 1 将提供 CRP 活性结构域及其结合的分子分析 CRP 与其配体（补体成分 C3b）之间的相互作用。这 将使用表达的突变CRP构建体进行功能分析 在哺乳动物细胞中检测补体限制活性和 C3b 绑定。此外，我们将使用中和性抗CRP单克隆抗体 和 CRP 结合肽以提供更精细的活性图谱。具体目标2 将重点在小鼠模型中评估基于 CRP-DNA 的疫苗 恰加斯病。 在这些研究中，我们将比较三种表达载体，优化 免疫方案，并使用多种方法评估保护范围 小鼠和寄生虫菌株。此外，我们还会检查口腔和粘膜 疫苗输送系统和粘膜亚致死攻击方案。目标 具体目标 3 是分析参与免疫效应的机制 保护性抗CRP反应。免疫抗体的作用将在以下方面进行评估： DNA 免疫小鼠以确定构象依赖性表位是否 对于诱导保护性反应至关重要。被动转移和 将进行挑战研究，我们将测试接种疫苗的小鼠 使用表达 CRP 的靶细胞检测 CRP 特异性细胞毒性 T 细胞的存在。 这些目标的成功完成将提供第一个功能性和 对克氏锥虫的关键毒力因子进行免疫学评估并提供 使用 CRP 作为候选疫苗的信息和试剂。