Accelerating discovery of neutralizing paratopes with Functional Antibody Screening Technology

利用功能性抗体筛选技术加速中和互补位的发现

基本信息

批准号：
10088379
负责人：
Arum Han
金额：
$ 18.94万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-24 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10088379
关键词：
Address Advanced Development Antibodies Antibody Binding Sites Antibody Diversity Antibody Repertoire B-Lymphocytes Biological Biological Assay Biological Models Biological Process Cells Cessation of life Communicable Diseases Consumption Data Dengue Virus Devices Environment Event Face Generations HIV Heterophile Antibodies Human Resources Hybridomas Immunization Immunoglobulin-Secreting Cells Immunology Individual Infection Infectious Agent Libraries Licensure Longevity Masks Methodology Methods Microfluidic Microchips Microfluidics Middle East Respiratory Syndrome Coronavirus Modeling Monoclonal Antibodies Murine hepatitis virus Mus Pain Performance Plasmablast Population Research Research Personnel Research Support Resolution Resources SARS coronavirus Serotherapies Source System Technology Testing Texas Therapeutic Therapeutic Intervention Therapeutic antibodies Time Vaccine Antigen Viral Viral Antigens Viral hepatitis Virus Virus Diseases Work Yeasts antibody libraries antibody test antigen antibody binding base cost design digital human coronavirus influenzavirus innovation laboratory facility male health nanofabrication neutralizing antibody new technology novel novel strategies pathogen pathogenic virus polyclonal antibody screening therapeutic vaccine therapeutically effective treatment response vaccine development virology

项目摘要

ABSTRACT Viral pathogens cause enormous pain and suffering, and more than 1B infections and 100M deaths each year. Unfortunately, safe and effective therapeutic interventions or vaccines are unavailable for many viral pathogens of global consequence. The identification of neutralizing antibodies (nAbs) against these pathogens would enable passive serotherapy as well as vaccine antigen (Ag) identification. However, determining the functional repertoire of nAbs elicited by viral infection faces two daunting challenges. First, several immunization- based methodologies enable antibody (Ab) discovery. These approaches reliably support the generation of polyclonal sera and, with hybridoma technology, renewable sources of monoclonal Abs. However, polyclonal Abs are non-renewable and hybridoma generation is costly, time-consuming, and results in a substantial loss of Ab diversity (i.e., biased). Direct screening of the vast repertoire of primary B cells is ideal for nAb discovery. However, primary plasmablast cultivation is challenging, and the limited lifespan of Ab- producing primary plasmablasts in culture makes direct screening of these cells impractical. Second, available assays for testing Ab function are low throughput, costly, and require enormous commitments of manpower and resources to execute. Here, an innovative method that can address all of these challenges is proposed. Heterologous Ab secretion by yeast cells that can be readily cultivated, and do not perturb viral infection of host cells, constitute an attractive alternative. This proposal will develop FAST (Functional Ab Screening Technology), a novel droplet microfluidics platform that combines high-throughput microfluidics and yeast Ab secretion library technology to rapidly identify nAbs against viruses, achieving dramatic increases in throughput and corresponding reductions in cost, compared to conventional systems. Importantly, because the biological functions of Abs expressed in yeast (viral neutralization) are directly tested in the system rather than simple Ag-Ab binding interactions, FAST is expected to return high-quality, biologically meaningful hits. To date, exciting proof-of concept data showing that the FAST system can be used to identify Abs that neutralize murine hepatitis virus (MHV) infection has been collected. To test the central hypothesis that FAST will deliver a rapid, low cost, unbiased, approach to identifying Abs that neutralize viral infection, this proposal will: AIM 1. To optimize FAST by executing a mock screen using a model system against MHV; AIM 2. To test the working hypothesis that an Ab library derived from MHV-infected mice contains potent MHV neutralizing molecules. If successful, this work will establish the first high-throughput system for performing functional viral neutralization bioassays at single-cell (digital) resolution, allowing identification of nAbs against viral pathogens of global consequence.

抽象的病毒病原体会造成巨大的痛苦和折磨，每年造成超过 1B 例感染和 1 亿人死亡。不幸的是，许多病毒无法获得安全有效的治疗干预措施或疫苗具有全球影响的病原体。针对这些病原体的中和抗体 (nAb) 的鉴定将使被动血清疗法以及疫苗抗原（Ag）识别成为可能。然而，确定病毒感染引起的 nAb 功能库面临着两个艰巨的挑战。首先，几个基于免疫的方法能够发现抗体 (Ab)。这些方法可靠地支持产生多克隆血清，并利用杂交瘤技术，获得单克隆抗体的可再生来源。然而，多克隆抗体是不可再生的，杂交瘤的产生成本高昂、耗时且结果不佳 Ab 多样性大幅丧失（即有偏差）。直接筛选大量原代 B 细胞非常适合 nAb 发现。然而，原代浆母细胞培养具有挑战性，并且 Ab- 的寿命有限。在培养物中产生原代浆母细胞使得直接筛选这些细胞变得不切实际。二、可用检测抗体功能的检测方法通量低、成本高且需要投入大量人力和执行资源。在这里，提出了一种可以解决所有这些挑战的创新方法。酵母细胞分泌的异源抗体易于培养，并且不会干扰病毒感染宿主细胞构成了一个有吸引力的替代方案。该提案将开发FAST（功能性抗体筛选技术），一种结合了高通量微流控和酵母抗体的新型液滴微流控平台分泌库技术可快速识别针对病毒的nAb，实现通量的大幅提高与传统系统相比，成本相应降低。重要的是，因为生物酵母中表达的抗体（病毒中和）的功能是直接在系统中测试的，而不是简单的 Ag-Ab 结合相互作用，FAST 有望返回高质量、具有生物学意义的命中结果。迄今为止，令人兴奋的概念验证数据表明 FAST 系统可用于识别中和抗体已收集小鼠肝炎病毒（MHV）感染。检验 FAST 将实现的中心假设一种快速、低成本、公正的方法来识别中和病毒感染的抗体，该提案将： AIM 1。通过使用针对 MHV 的模型系统执行模拟屏幕来优化 FAST；目的 2. 测试工作假设来自 MHV 感染小鼠的抗体库含有有效的 MHV 中和分子。如果如果成功，这项工作将建立第一个用于执行功能性病毒中和的高通量系统单细胞（数字）分辨率的生物测定，可识别针对全球病毒病原体的 nAb 结果。