Heat Stable Filoviral Diagnostics

描述（由申请方提供）：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检测性能的分子要求。这两种丝状病毒继续在非洲造成毁灭性的出血热暴发，并通过受感染旅行者的返回、受感染的非人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。缺乏疫苗和有效的治疗手段意味着快速诊断对于控制疫情至关重要，无论疫情发生在哪里。目前的诊断在耐用性、便携性和可用性方面受到限制，特别是在资源匮乏的环境中，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，精心选择的美洲驼单域抗体（sdAb）蛋白特异性的五个丝状病毒物种的每一个将使组装快速，准确，但强大的诊断。我们基于我们的研究，这一假设：1。与常规免疫球蛋白不同，针对不同抗原选择的sdAb在暴露于高温时保留特异性抗原结合活性，和2.选择针对马尔堡病毒的sdAb使得能够建立快速抗原捕获测定，其在灵敏度和特异性方面匹配基于核酸的检测方法。我们的具体目标是：1.表征已经针对马尔堡病毒选择的预先存在的sdAb的热稳定性和分子识别的要求; 2.表征在扎伊尔埃博拉病毒、苏丹埃博拉病毒、科特迪瓦埃博拉病毒和雷斯顿埃博拉病毒上选择的sdAb，以确定测定灵敏度-特异性的程度是否保守以及sdAb是否是热稳定的; 3.开始合理化埃博拉sdAb的分子识别，并结合马尔堡sdAb数据，了解有效抗原捕获检测开发的规则和要求。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具有潜在的无限保质期。公共卫生相关性：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暴发。其他国家也因输入受感染的非人灵长类动物、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。由于目前没有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感染者并对其进行隔离，以减少疾病传播。我们的目标是开发新型、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能够等于基于核酸的检测限的机制，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.