Heat Stable Filoviral Diagnostics

描述(由申请人提供)：这项建议的广泛长期目标是开发马尔堡病毒和埃博拉病毒的热稳定免疫分析试剂，并开始阐明有效的抗原捕获分析性能的分子要求。这两种丝状病毒继续在非洲造成毁灭性的出血热暴发，并通过受感染的旅行者返回、受感染的非人类灵长类动物的输入和生物恐怖主义的威胁，威胁到非地方病国家。缺乏疫苗和有效的治疗方法意味着，无论在哪里爆发，快速诊断对于遏制疫情都是至关重要的。目前的诊断在耐用性、便携性和可用性方面都是有限的，特别是在资源匮乏的情况下，我们的目标是增强这些能力，以帮助保障公众健康。我们假设，精心挑选的针对五种丝状病毒每种的骆驼单域抗体(SdAb)蛋白将使快速、准确而稳健的诊断组装成为可能。我们基于我们的研究假设：1.与常规免疫球蛋白不同，选择不同抗原的sdAbb在高温下保持特定的抗原结合活性；2.选择Marburg病毒sdAbb可以建立在灵敏度和特异性方面与核酸检测方法相匹配的快速抗原捕获检测方法。我们的具体目标是：1.对已有的针对马尔堡病毒的sdAb的热稳定性和分子识别要求进行表征；2.对在埃博拉、扎伊尔、苏丹、象牙海岸和雷斯顿选择的sdAb进行表征，以确定检测的敏感性-特异性程度是否保守，以及sdAb是否具有热稳定性；3.开始合理化埃博拉sdAb的分子识别，并结合马尔堡sdAb的数据，了解有效的抗原捕获试验发展的规则和要求。这些研究将指导人们开发出廉价、简单但超敏感的丝状病毒诊断方法，即使在没有冷藏的情况下，也可能有无限的保质期。与公共卫生相关：丝状病毒马尔堡和埃博拉继续在非洲造成不可预测的传染性和高致命性出血热的暴发。其他国家也因进口受感染的非人类灵长类动物、受感染的游客从流行地区返回以及生物恐怖主义的威胁而面临风险。由于目前还没有针对这些病毒的疫苗或治疗方法，因此必须在现场快速识别感染者并对他们进行隔离，以减少疾病传播。我们的目标是开发针对丝状病毒物种的新的、持久的抗体，以帮助开发廉价、快速的诊断测试，以满足这一迫切需求。我们还希望研究这些新的抗丝状病毒抗体如何达到基于核酸的检测极限的机制，以了解如何制定改进的病毒抗原捕获试验。

DESCRIPTION (provided by applicant): The broad long-term objectives of this proposal are to develop heat stable immunoassay reagents for Marburg and Ebola viruses and begin elucidating the molecular requirements for effective antigen capture assay performance. Both filoviruses continue to cause devastating outbreaks of hemorrhagic fever in Africa and threaten non-endemic countries, through the return of infected travelers, the importation of infected non-human primates and the threat of bioterrorism. The absence of vaccines and effective therapeutics means that rapid diagnosis is essential to contain an outbreak wherever it may occur. Current diagnostics are limited in terms of durability, portability and availability especially in resource poor settings and our aim is to augment these capabilities to help safeguard public health. We hypothesize that carefully chosen llama single domain antibody (sdAb) proteins specific for each of the five filovirus species will enable the assembly of rapid, accurate yet robust diagnostics. We base this hypothesis upon our studies that: 1. sdAbs selected to diverse antigens retain specific antigen binding activity when exposed to high temperatures unlike conventional immunoglobulins and 2. sdAbs selected to Marburg virus enable rapid antigen capture assays to be established that match nucleic acid based detection methods in terms of sensitivity and specificity. Our specific aims are to: 1. Characterize pre-existing sdAbs that have been selected against Marburg virus for their thermostability and requirements for molecular recognition; 2. Characterize sdAbs selected on Ebola Zaire, Sudan, Ivory Coast and Reston to determine if the degree of assay sensitivity-specificity is conserved and if the sdAbs are thermostable; 3. Begin to rationalize the molecular recognition of the Ebola sdAb and, in concert with the Marburg sdAb data, understand the rules and requirements for effective antigen capture assay development. These studies will guide the way to formulating inexpensive, simple yet ultrasensitive filoviral diagnostics with potentially infinite shelf lives even in the absence of refrigeration. PUBLIC HEALTH RELEVANCE: The filoviruses Marburg and Ebola continue to cause unpredictable outbreaks of transmissible and highly lethal hemorrhagic fevers in Africa. Other countries are also at risk through the importation of infected non-human primates, the return of infected tourists from endemic regions, and the threat of bioterrorism. Since no vaccines or therapeutics are currently available for these viruses, it is imperative to rapidly identify infected individuals on-site and quarantine them to reduce disease transmission. We aim to develop novel, durable antibodies specific for filoviral species to aid the development of inexpensive, rapid diagnostic tests to meet this urgent need. We also wish to study the mechanism of how these novel anti-filoviral antibodies can equal nucleic acid based detection limits to understand how to formulate improved virus antigen capture assays.