High Throughput Analysis of Malarial Surface Antigens

项目描述（由申请人提供）：本项目的主要目标是开发阵列技术，研究恶性疟原虫表面蛋白的抗原性和功能，恶性疟原虫是感染人类的最致命的疟疾物种。变异表面蛋白PfEMPI是恶性疟原虫的关键毒力分子，介导寄生虫感染的红细胞粘附和抗原变异，不同形式的PfEMPI与严重疟疾和妊娠疟疾的发病机制有关。尽管其在疾病和保护中发挥关键作用，但由于其复杂性、大尺寸和表达困难，对PfEMPI变异体的研究受到限制。恶性疟原虫3D7株基因组测序完成，为高通量技术应用于所有感兴趣的基因产物提供了基础。我们将克隆菌株3D7中编码PfEMPI的59个基因的所有组成域，以及其他现有的疫苗候选抗原。我们将确定最有效的方法来表达和保存这些抗原的抗体反应性，使用无细胞翻译和细胞表面表达。重组抗原的抗体反应性将在天然和变性条件下进行验证，使用从免疫血清中自然获得的抗体以及构象敏感的单克隆抗体。抗原阵列将通过自组装或点阵技术构建表达蛋白，并与原位细胞转染或细胞点阵技术在玻片表面进行比较。试点研究将用坦桑尼亚纵向队列研究中收集的血清样本测试抗原阵列，这些研究将证明蛋白质阵列平台在未来用于更大规模的疟疾免疫流行病学研究的可行性。此外，重组抗原的构象也将通过排列的PfEMPI结构域在内皮受体分子粘附实验中得到验证。这些实验也可以确定额外的PfEMPI结构域结合特性，可以在未来的研究中详细研究。从这些试验中获得的免疫特征或抗体谱可以提供一种快速、有效和系统的方法来确定疫苗的候选抗原。未来，蛋白质阵列可以扩展到整个蛋白质组，并用于功能研究，包括粘附，蛋白质-蛋白质相互作用，以及针对多态抗原的变异特异性抗体的获取。

DESCRIPTION (provided by the applicant): The broad objective of this project is to develop array technologies to study the antigenicity and function of surface proteins of Plasmodium falciparum, the most virulent malaria species infecting humans. The variant surface protein PfEMPI is a key virulence molecule of P. falciparum that mediates parasite-infected erythrocyte adhesion as well as antigenic variation, and distinct forms of PfEMPI have been implicated in the pathogenesis of severe malaria and pregnancy malaria. Despite its key role in disease and protection, studies of PfEMPI variants are limited owing to its complexity, large size, and difficulty in expression. Sequencing of P. falciparum strain 3D7 genome is complete, and provides the basis for applying high throughput technologies to all gene products of interest. We will clone all constituent domains of 59 genes encoding PfEMPI in strain 3D7, as well as other current vaccine candidate antigens. We will determine the most efficient method for expression and preservation of antibody reactivity of these antigens, using cell-free translation and cell-surface expression. Antibody reactivity of recombinant antigens will be validated under native and denaturing conditions, using naturally acquired antibody from immune sera as well as conformation-sensitive monoclonal antibodies. Antigen arrays will be constructed using expressed proteins arrayed by self-assembling or spotting technologies, and compared with in situ cell-transfection or cell- spotting technologies on the surface of glass slides. Pilot studies will test the antigen arrays with serum samples collected in a longitudinal cohort study in Tanzania, and these will demonstrate the feasibility of protein array platforms for larger malaria immuno-epidemiology studies in the future. In addition, the conformation of recombinant antigens will also be validated in adhesion experiments with endothelial receptor molecules using arrayed PfEMPI domains. These experiments may also identify additional PfEMPI domain binding properties that could be investigated in detail in future studies. Immunological signatures or antibody profiles obtained from these assays may provide a rapid, efficient and systematic approach to identify candidate antigens for vaccines. In future, protein arrays can be expanded to the entire proteome, and used for functional studies including adhesion, protein-protein interactions, and variant-specific acquisition of antibodies against polymorphic antigens.