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，通常是致命的病毒性出血热(VHF)。由于其高病死率、容易通过人与人接触传播以及可能释放气溶胶，拉沙热的病原体拉萨病毒(LASV)被归类为生物安全4级和NIAID生物防御A类制剂。我们的团队已经成功地生产出基于重组蛋白而不是必须在高密封性实验室生产的试剂的LASV酶联免疫吸附分析(ELISA)原型。我们还在塞拉利昂建立了一个研究计划，为VHF研究提供独特的临床和实验室资源。塞拉利昂是LASV的流行区。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。在里程碑1中，我们将开发商业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，使用一个具有良好特性的血清的性能小组。在里程碑2中，我们将开发拉沙热重组横向流动点诊断学。在里程碑3中，我们将在质量保证(QA)/质量控制(QC)的良好制造规范(GMP)下转换为生产重组ELISA和侧向流动分析，以提供大量足以用于设计控制参数临床前评估的商业级诊断试剂盒，以达到FDA批准所需的基准。在里程碑4中，我们将优化重组LASV蛋白、GP1、GP2和NP以及针对重组LASV GP1、GP2和NP的单抗的放大/纯化。我们还将改用QA/QC生产，以提供大量足以开发和测试商业检测的重组蛋白和单抗。在里程碑5中，我们将定义和收集来自西非和其他地区拉沙热流行范围内不同地区的阳性和阴性血清，以进行化验验证。这些血清将用于商业LASV重组ELISA法和重组横向流动点诊断试验，并与BSL-4EL ISA和PCR法的结果进行比较。相关性(见说明)：LASV作为生物武器的潜在用途需要开发诊断暴露于LASV和/或感染LASV的个人的方法。拉沙热在西非流行地区的影响是巨大的，安全和有效的诊断也可以提供非常显著的公共卫生惠益。

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.