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

项目摘要/摘要 疟疾每年感染2亿多人，并导致40多万儿童死亡。大多数感染是由 通过快速诊断试验(RDT)检测到恶性疟原虫，从而使临床能够可靠地 在现场环境中进行诊断。然而，在多个环境中，耐诊断的恶性疟原虫有 出现了不表达大多数检测所针对的抗原的富含组氨酸蛋白2(HRP2)。这些 HRP2缺失的寄生虫会导致假阴性的RDT结果，这既阻碍了适当的治疗，也阻碍了 削弱对RDT指导的临床疟疾治疗的投资。出于这个原因，我们迫切需要 识别恶性疟原虫的新诊断靶点，并开发能够特异性检测恶性疟原虫的诊断试剂 这些目标具有很高的敏感性。不幸的是，确定特定疾病的最佳诊断目标 病原体已经被证明是困难的。大多数诊断目标都是通过反复试验确定的。在这里，我们 建议使用我们开发的一种新的高通量筛选程序来鉴定新的 恶性疟原虫的诊断靶点。这一过程是基于使用单链抗体(ScFv)噬菌体 显示，旨在快速识别新的诊断目标和高亲和力捕获和检测 针对这些靶点的抗体。该程序的优点是速度快，缩短了时间 识别诊断靶点和诊断捕获/检测抗体对所需 几年到几个月，它的保真度，特别是识别抗体组合将提供 在RDT中部署时具有最高的临床敏感性。我们的建议包括三个具体目标。首先，我们将 利用新型噬菌体载体构建恶性疟原虫特异性免疫单链抗体噬菌体展示文库 更快速地筛选和鉴定单链抗体克隆。第二，我们将应用我们的小说放映 用于确定最佳恶性疟原虫诊断靶点和诊断抗体对的程序 特定于这些目标。第三，我们将确定那些最有潜力的诊断抗体对 根据对多重P。的敏感性和广度诊断疟疾的临床实用价值。 恶性疟原虫分离株。这些研究的成功完成将导致小说的鉴定 恶性疟原虫的诊断靶点，并提供针对这些靶点的有效诊断抗体，这 可用于检测HRP2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 新的筛查程序是识别新的诊断靶点和抗体的有效手段， 然后可以应用于准确的快速诊断不能用于广泛的传染病 目前存在的原因是缺乏经过验证的诊断目标。

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.