Generation of antibodies specific for optimal non-HRP2 malaria diagnostic antigens

项目概要/摘要 每年有2亿多人感染疟疾，40多万儿童死亡。大多数感染是由 通过快速诊断试验（RDT）检测恶性疟原虫，这使得可靠的临床诊断成为可能。 现场诊断。然而，在多种情况下，“耐诊断”恶性疟原虫寄生虫 出现了不表达大多数测试靶向的抗原，富组氨酸蛋白2（HRP 2）。这些 HRP 2缺失的寄生虫导致假阴性RDT结果，这既妨碍了适当的治疗， 损害了对快速诊断疗法指导下的诊所疟疾治疗的投资。因此，迫切需要 确定恶性疟原虫的新诊断靶点，并开发特异性检测恶性疟原虫的诊断试剂， 这些目标具有高灵敏度。不幸的是，识别特定的最佳诊断靶点， 病原体已经被证明是困难的。大多数诊断目标是通过反复试验确定的。这里我们 我建议使用一种新的高通量筛选程序，我们已经开发，以确定新的 恶性疟原虫的诊断目标。该方法基于单链抗体（scFv）噬菌体的使用， 显示，并被设计为快速识别新的诊断目标和高亲和力的捕获和检测 针对这些目标的抗体。该程序的优点包括其速度，减少时间 这是鉴定诊断靶标和诊断捕获/检测抗体对所需的， 几年到几个月，以及它的保真度，特别是识别抗体组合，将提供 在RDT中展开时的最大临床灵敏度。我们的建议包括三个具体目标。一是 使用新的噬菌粒载体产生恶性疟原虫特异性免疫scFv噬菌体展示文库， 更快速地筛选和表征scFv克隆。第二，我们将应用我们的小说筛选 鉴定最佳恶性疟原虫诊断靶标和诊断抗体对的方法 针对这些目标。第三，我们将确定那些具有最大潜力的诊断抗体对 在对多种P. 恶性疟原虫分离株。这些研究的成功完成将导致确定新的 恶性疟原虫的诊断靶点，并提供对这些靶点特异性的经验证的诊断抗体， 可用于检测HRP 2缺失的恶性疟原虫菌株的感染。此外，这项工作将验证我们的 新的筛选程序作为鉴定新的诊断靶标和抗体的有效手段， 然后可以应用于广泛的传染病，对于这些传染病， 目前由于缺乏有效的诊断目标而存在。

Project Summary/Abstract Malaria infects over 200 million people and kills over 400,000 children annually. Most infections result from Plasmodium falciparum and are detected by rapid diagnostic tests (RDTs), which enable reliable clinical diagnosis in field settings. However, in multiple settings “diagnosis-resistant” P. falciparum parasites have emerged that do not express the antigen targeted by most tests, Histidine-Rich Protein 2 (HRP2). These HRP2-deleted parasites result in false-negative RDT results, which both prevent proper treatment and undermine investments in RDT-guided malaria treatment in the clinic. For this reason, there is a critical need to identify new diagnostic targets for P. falciparum and develop diagnostic reagents that will specifically detect these targets with high sensitivity. Unfortunately, the identification of optimal diagnostic targets for specific pathogens has proven difficult. Most diagnostic targets have been identified by trial and error. Here, we propose to use a novel high throughput screening procedure that we have developed to identify novel diagnostic targets for P. falciparum. This procedure is based on the use of single-chain antibody (scFv) phage display and is designed to rapidly identify both novel diagnostic targets and high-affinity capture and detection antibodies specific for these targets. Advantages of this procedure include its speed, reducing the time required for the identification of both diagnostic targets and diagnostic capture/detection antibody pairs from several years to a few months, and its fidelity, specifically identifying antibody combinations that will provide the greatest clinical sensitivity when deployed in RDTs. Our proposal includes 3 Specific Aims. First, we will generate a P. falciparum-specific immune scFv phage display library using a novel phagemid vector that allows a much more rapid screening and characterization of scFv clones. Second, we will apply our novel screening procedure to this library to identify the optimal P. falciparum diagnostic targets and diagnostic antibody pairs specific to these targets. Third, we will identify those diagnostic antibody pairs that have the greatest potential clinical utility for the diagnosis of malaria in terms of sensitivity and breadth of reactivity against multiple P. falciparum isolates. The successful completion of these studies will result in the identification of novel diagnostic targets for P. falciparum and provide validated diagnostic antibodies specific for these targets, which can be used to detect infection by HRP2-deleted P. falciparum strains. In addition, this work will validate our novel screening procedure as an effective means to identify novel diagnostic targets and antibodies, which may then be applied to a wide range of infectious diseases for which accurate rapid diagnostics do not currently exist due to a lack of validated diagnostic targets.