Mechanism and Evolution of Filoviral Monoclonal Affinity Reagent Sandwich Assays

 描述（由申请方提供）：快速检测病毒特征对于诊断丝状病毒病以加快隔离、限制传染病传播和遏制疫情爆发非常重要。在爆发环境中有效和经济地实现这一点并不是微不足道的，特别是当病毒数量在感染过程的早期很低时。使用对核蛋白（NP）的C末端具有特异性的美洲驼单域抗体（sdAb），我们能够检测到低滴度的马尔堡和埃博拉病毒，尽管尚未达到qRT-PCR可达到的水平。每种sdAb能够在抗原捕获测定中充当捕获物和示踪物，依赖于与多价抗原的亲合力或Velcro样效应。抗马尔堡sdAb-NP复合物的结构分析已经确定了表位，并表明它们在自1967年首次出现以来记录的所有病毒株中是保守的。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，开发持久的免疫测定法，不仅匹配基于核酸的检测灵敏度，而且具有识别和分类丝状病毒株的高概率。我们将通过三个具体目标实现这一目标：SA 1，简化测定格式，因为我们假设可以简化现有测定，而不会从4步捕获到3步捕获和新型1步溶液相测定失去灵敏度或特异性。我们将针对qRT-PCR对我们的新免疫测定的灵敏度、特异性、重现性和耐久性进行基准测试; SA 2，提高马尔堡病毒测定灵敏度，因为我们假设我们的sdAb具有未优化的NP识别机制，可以进行微调以提高检测限，同时保留马尔堡病毒株交叉反应性。将使用结构导向设计来进化sdAb互补位，确保任何改变的表位仍然是保守的，并且将功能突变体的获得馈送到SA 1的简化测定和基准中; SA3，调节埃博拉病毒测定交叉反应性，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，反应性sdAb被工程化，但具有足够的多样性以产生物种特异性sdAb。部分交叉反应性sdAb的结构将被解析，并且信息用于指导广泛交叉反应性sdAb的进化，而聚NP免疫文库的消减选择将用于产生物种特异性sdAb。将检查新sdAb的表位，以确保它们在相关物种中完全保守，并如SA 1中那样用于精简和基准测试。开创性的简单、快速和廉价的基于sdAb的丝状病毒免疫测定，匹配qRT-PCR的灵敏度和特异性，将通过为疫情环境提供全面的诊断工具包来帮助保护人类健康。创新的方法和新的见解，免疫分析组装将加快诊断对策，对其他传染病的代理人，特别是快速演变的新兴负链RNA病毒与多种血清型。

 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.