Mechanism and Evolution of Filoviral Monoclonal Affinity Reagent Sandwich Assays

 描述(由申请人提供)：快速检测病毒特征对于诊断丝状病毒病、加快隔离、限制传染病传播和遏制疫情非常重要。在暴发环境中高效和经济地实现这一点并非易事，特别是在感染过程早期病毒数量较低的情况下。使用针对核蛋白(NP)C末端的骆驼单域抗体(SdAb)，我们能够检测到低滴度的马尔堡病毒和埃博拉病毒，尽管还不能达到qRT-PCR所能达到的水平。每个sdAb在抗原捕获试验中既可以作为捕获者又可以作为示踪剂，依赖于多价抗原的亲和力或尼龙搭扣效应。对抗Marburg sdAb-NP复合体的结构分析确定了表位，并表明自1967年首次出现以来，它们在所有记录的病毒株中都是保守的。我们广泛的长期目标是利用sdAb和高度保守的丝状病毒NP表位之间相互作用的知识，开发持久的免疫分析方法，不仅匹配基于核酸的检测灵敏度，而且有很高的可能性识别和分类出现的丝状病毒YE株。我们将通过三个具体目标来实现这一点：SA1，简化分析格式，因为我们假设我们现有的分析可以简化，而不会损失灵敏度或特异性，从4步捕获到3步捕获和新的1步溶液相分析。我们将针对qRT-PCR对我们新的免疫分析方法的灵敏度、特异性、重复性和持久性进行基准测试；SA2，提高马尔堡病毒分析的灵敏度，因为我们假设我们的sdAb具有未优化的NP识别机制，可以对其进行微调，以提高检测限，同时保持马尔堡病毒株的交叉反应。结构导引设计将被用于进化sdAb副表位，确保任何改变的表位仍然是保守的，并获得功能突变被馈入简化的分析和基准SA1；SA3，调节埃博拉病毒的交叉反应，因为我们假设五个埃博拉病毒NP C末端结构域具有足够的表位保守性，使能够设计出泛反应性的sdAb，同时也有足够的多样性来产生物种特异性的sdAb。部分交叉反应sdAb的结构将被解决，用于指导广泛交叉反应sdAb进化的信息将被用于指导，而多NP免疫库的消减选择将被用于产生物种特异性sdAb。将检查新的sdAb的表位，以确保它们在相关物种中完全保守，并像SA1一样被纳入精简和基准。开创了基于简单、快速和廉价的丝状病毒免疫分析的先河，与qRT-PCR的敏感性和特异性相匹配，将通过为暴发环境提供全面的诊断工具包来帮助保护人类健康。对免疫分析组装的创新方法和新见解将加快对其他传染病病原体的诊断对策，特别是快速进化的具有多种血清型的负链RNA病毒。

 DESCRIPTION (provided by applicant): Rapidly detecting viral signatures is important in diagnosing Filovirus disease to hasten quarantine, limit the spread of contagion, and contain an outbreak. Achieving this efficiently and economically in an outbreak setting is not trivial, especially when virus numbers are low early in the course of infection. Using llama single domain antibodies (sdAb) specific for the C-terminus of nucleoprotein (NP), we are able to detect low titers of both Marburg and Ebola viruses though not yet at the levels achievable by qRT-PCR. Each sdAb is able to act as both captor and tracer in an antigen capture assay, relying avidity or Velcro-like effect with polyvalent antigen. Structural analysis of the anti-Marburg sdAb-NP complexes has defined the epitopes and shown that they are conserved among all virus strains documented since the first emergence in 1967. Our broad long term goal is to leverage knowledge of the interaction between sdAb and highly conserved Filoviral NP epitopes, to develop durable immunoassays that not only match nucleic acid based detection sensitivities but will have a high probability of recognizing and categorizing Filoviral strains ye to emerge. We will achieve this with three specific aims: SA1, simplify the assay format as we hypothesize our existing assays can be streamlined without losing sensitivity or specificity from a 4 step to a 3 step capture and novel 1 step solution phase assay. We will benchmark the sensitivity, specificity, reproducibility and durability of our new immunoassays against qRT-PCR; SA2, improve Marburg virus assay sensitivity as we hypothesize our sdAb have un-optimized NP recognition mechanisms that can be fine-tuned to improve limits of detection while retaining Marburg virus strain cross-reactivity. Structure guided design will be used to evolve sdAb paratopes, ensure any altered epitopes are still conserved, and gain of function mutants fed into the streamlined assays and benchmarking of SA1; SA3, modulate Ebola virus assay cross-reactivity as we hypothesize the five Ebola virus NP C-terminal domains have sufficient epitope conservation to enable a pan-reactive sdAb to be engineered yet enough diversity for species specific sdAb to be generated. The structure of a partially cross- reactive sdAb will be solved and the information used to guide the evolution of a broadly cross-reactive sdAb, while subtractive selection of a poly-NP immune library will be used to generate species specific sdAb. Epitopes of the new sdAb will be checked to ensure they are completely conserved among the relevant species and fed into streamlining and benchmarking as in SA1. Pioneering simple, fast and inexpensive sdAb based Filoviral immunoassays that match qRT-PCR sensitivity and specificity, will help safeguard human health by providing a comprehensive diagnostic toolkit for outbreak settings. The innovative approaches and new insights into immunoassay assembly will accelerate diagnostic countermeasures against other infectious disease agents, particularly rapidly evolving emerging negative strand RNA viruses with multiple serotypes.