Heat Stable Filoviral Diagnostics

描述（由申请人提供）：本提案的广泛长期目标是开发用于马尔堡和埃博拉病毒的热稳定免疫测定试剂，并开始阐明有效抗原捕获测定性能的分子要求。这两种丝状病毒继续在非洲引起毁灭性的出血热暴发，并通过受感染旅行者的返回、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非流行国家。由于缺乏疫苗和有效的治疗方法，快速诊断对于控制任何可能发生的疫情至关重要。目前的诊断方法在持久性、可移植性和可用性方面有限，特别是在资源贫乏的环境中，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，精心选择的骆驼单域抗体（sdAb）蛋白对五种线状病毒的每一种都有特异性，将使快速、准确且可靠的诊断成为可能。我们基于以下研究得出这一假设：与常规免疫球蛋白和免疫球蛋白不同，选择不同抗原的单克隆抗体在高温下保持特异性抗原结合活性。选择针对马尔堡病毒的单克隆抗体，可以建立快速抗原捕获测定方法，在敏感性和特异性方面与基于核酸的检测方法相匹配。我们的具体目标是：1。描述针对马尔堡病毒选择的已有单克隆抗体的热稳定性和分子识别要求；2. 对在扎伊尔、苏丹、科特迪瓦和雷斯顿选取的单克隆抗体进行表征，以确定检测敏感性特异性是否保守，单克隆抗体是否耐热；3. 开始使埃博拉sdAb的分子识别合理化，并结合马尔堡sdAb数据，了解有效抗原捕获测定开发的规则和要求。这些研究将指导制定廉价，简单但超灵敏的丝状病毒诊断方法，即使在没有冷藏的情况下也可能具有无限的保质期。公共卫生相关性：马尔堡和埃博拉丝状病毒继续在非洲引起不可预测的传染性和高致命性出血热暴发。由于输入受感染的非人类灵长类动物、从流行地区返回受感染的游客以及生物恐怖主义的威胁，其他国家也面临风险。由于目前尚无针对这些病毒的疫苗或治疗方法，因此必须在现场迅速识别受感染个体并对其进行隔离，以减少疾病传播。我们的目标是开发新的、持久的丝状病毒特异性抗体，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型抗丝状病毒抗体如何与基于核酸的检测限相等的机制，以了解如何制定改进的病毒抗原捕获测定法。

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.