Preclinical development of recombinant antigen diagnostics for Lassa fever

描述（由申请人提供）：该项目将开发针对拉沙热的生物防御诊断，拉沙热是一种严重的，通常致命的病毒性出血热（VHF）。由于其高病死率，通过人与人接触容易传播的能力，以及气溶胶释放的潜力，拉沙病毒（LASV），拉沙热的病原体，被列为生物安全4级和NIAID生物防御A类制剂。我们的团队已经成功地生产了原型LASV酶联免疫吸附测定（ELISA），它是基于重组蛋白，而不是必须在高遏制实验室生产的试剂。我们还在塞拉利昂（LASV流行地区）新设立了一个研究项目，为VHF研究提供独特的临床和实验室资源。我们现在将在商业重组抗原拉沙热诊断的临床前开发中执行关键步骤。在MILESTONE 1中，我们将使用充分表征的血清的Performance Panel开发商业化拉沙热重组抗原捕获和免疫球蛋白M（IgM）和免疫球蛋白G（IgG）抗体捕获ELISA。在MILESTONE 2中，我们将开发拉沙热重组侧流床旁诊断。在MILESTONE 3中，我们将根据具有质量保证（QA）/质量控制（QC）的药品生产质量管理规范（GMP）生产重组ELISA和侧流检测试剂盒，以提供足够数量的商业级诊断试剂盒，用于设计控制参数的临床前评价，以达到FDA批准所需的基准。在MILESTONE 4中，我们将优化重组LASV蛋白、GP 1、GP 2和NP以及重组LASV GP 1、GP 2和NP的单克隆抗体（mAb）的放大/纯化。我们还将转换为具有QA/QC的生产，以提供足以用于商业测定的开发和检测的重组蛋白和mAb的数量。在MILESTONE 5中，我们将定义并收集来自西非和其他地方拉沙热流行范围内不同地区的阳性和阴性血清用于检测验证。这些血清将用于检测和比较商业LASV重组ELISA和重组侧流床旁诊断检测试剂与BSL-4 ELISA和PCR检测试剂的结果。相关性（参见说明）：LASV作为生物武器的潜在用途需要开发诊断暴露于和/或感染LASV的个体的方法。拉沙热在西非流行地区的影响是巨大的，安全有效的诊断也可以提供非常重要的公共卫生效益。

DESCRIPTION (provided by applicant): This project will develop Diagnostics For Biodefense against Lassa fever, a severe, often fatal viral hemorrhagic fever (VHF). Because of its high case fatality rate, ability to spread easily by human-human contact, and potential for aerosol release, Lassa virus (LASV), the causative agent of Lassa fever, is classified as a Biosafety Level 4 and NIAID Biodefense category A agent. Our team has successfully produced prototype LASV enzyme-linked immunosorbent assays (ELISA) that are based on recombinant proteins rather than on reagents that must be produced in high containment laboratories. We have also newly established a research program in Sierra Leone, an area endemic for LASV, that provides unique clinical and laboratory resources for VHF research. We will now perform critical steps in the preclinical development of commercial recombinant antigen Lassa fever diagnostics. In MILESTONE 1 we will development commercial Lassa fever recombinant antigen-capture and immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody-capture ELISA using a Performance Panel of well-characterized sera. In MILESTONE 2 we will development Lassa fever recombinant lateral flow point-of-care diagnostics. In MILESTONE 3 we will convert to manufacturing recombinant ELISA and lateral flow assays under Good Manufacturing Practices (GMP) with Quality Assurance (QA)/Quality Control (QC) to provide quantities of commercial grade diagnostic kits sufficient for preclinical evaluation of design control parameters to achieve benchmarks required for FDA approval. In MILESTONE 4 we will optimize scale up/purification of recombinant LASV proteins, GP1, GP2, and NP and monoclonal antibodies (mAbs) to recombinant LASV GP1, GP2, and NP. We will also convert to manufacturing with QA/QC, to provide quantities of recombinant proteins and mAbs sufficient for development and testing of commercial assays. In MILESTONE 5 we will define and collect positive and negative sera for assay validation from diverse regions across the Lassa fever endemic range of West Africa and elsewhere. These sera will be used to test and compare the commercial LASV recombinant ELISA and recombinant lateral flow point-of-care diagnostic assays with results from BSL-4 ELISA and PCR assays. RELEVANCE (See instructions): The potential use of LASV as a biological weapon necessitates development of methods to diagnose individuals exposed to and/or infected with LASV. The impact of Lassa fever in endemic areas of West Africa is immense, and a safe and effective diagnostic can also provide a very significant public health benefit.