High Throughput Analysis of Malarial Surface Antigens

描述（由申请人提供）：该项目的主要目标是开发阵列技术，以研究恶性疟原虫（感染人类的最致命疟疾物种）表面蛋白的抗原性和功能。PfEMPI是恶性疟原虫的一种重要毒力分子，介导疟原虫感染的红细胞粘附和抗原变异，不同形式的PfEMPI参与了重症疟疾和妊娠期疟疾的发病机制。尽管PfEMPI在疾病和保护中起着关键作用，但由于其复杂性、大尺寸和表达困难，PfEMPI变体的研究受到限制。恶性疟原虫菌株3D 7基因组的测序是完整的，并且为将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3D 7中编码PfEMPI的59个基因的所有组成结构域，以及其他当前的疫苗候选抗原。我们将使用无细胞翻译和细胞表面表达来确定表达和保存这些抗原的抗体反应性的最有效方法。重组抗原的抗体反应性将在天然和变性条件下使用来自免疫血清的天然获得性抗体以及构象敏感性单克隆抗体进行验证。将使用通过自组装或点样技术排列的表达蛋白构建抗原阵列，并与载玻片表面上的原位细胞转染或细胞点样技术进行比较。试点研究将用在坦桑尼亚的一项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用于未来更大规模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在使用阵列PfEMPI结构域与内皮受体分子的粘附实验中进行验证。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。从这些测定中获得的免疫学特征或抗体谱可以提供快速、有效和系统的方法来鉴定疫苗的候选抗原。将来，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，以及针对多态性抗原的抗体的变体特异性获取。

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.