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

• 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
• 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

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 病毒病原体会引起巨大的疼痛和痛苦，每年超过1B的感染和100m死亡。 不幸的是，对于许多病毒而无法获得安全有效的治疗干预措施或疫苗 全球后果的病原体。针对这些病原体的中和抗体（NAB）的鉴定 将实现被动血清疗法以及疫苗抗原（AG）鉴定。但是，确定 病毒感染引起的NABS功能曲目面临两个艰巨的挑战。首先，几个 基于免疫的方法可以使抗体（AB）发现。这些方法可靠地支持 多克隆血清的产生和杂交瘤技术，是单克隆ABS的可再生能源。 但是，多克隆ABS是不可再生的，杂交瘤的产生昂贵，耗时，结果 AB多样性的实质性丧失（即有偏见）。直接筛选主要B细胞的广泛曲目是 NAB发现的理想选择。然而，原发性浆膜种植具有挑战性，Ab-的寿命有限 在培养物中产生原发性浆液性使这些细胞直接筛选不切实际。第二，可用 测试AB功能的测定是低通量，昂贵的，并且需要巨大的人力承诺 和要执行的资源。在这里，提出了一种可以解决所有这些挑战的创新方法。 酵母细胞的异源AB分泌，可以容易培养，并且不会扰动病毒感染 宿主细胞是一种有吸引力的替代方法。该建议将快速发展（功能性AB筛查 技术），一种新型的液滴微流体平台，结合了高通量微流体和酵母AB 分泌图书馆技术快速识别针对病毒的NAB，实现吞吐量的急剧增加 与常规系统相比，成本的相应降低。重要的是，因为生物学 在酵母中表达的ABS的功能（病毒中和）在系统中直接测试，而不是简单 AG-AB结合相互作用，预计FAST将返回高质量的生物学上有意义的命中。迄今为止， 激动人心的概念验证验证数据表明，快速系统可用于识别中和的ABS 已收集了鼠肝炎病毒（MHV）感染。测试中心假设，即FAST将提供 快速，低成本，公正的方法来识别ABS中和病毒感染的ABS，该建议将：AIM 1。 通过使用MHV模型系统执行模拟屏幕来快速优化；目标2。测试工作 假设源自MHV感染的小鼠的AB文库包含有效的MHV中和分子。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，允许鉴定NABS针对全球病毒病原体 结果。