Accelerating discovery of neutralizing paratopes with Functional Antibody Screening Technology

摘要 病毒病原体造成巨大的痛苦和折磨，每年有超过1B的感染和1亿人死亡。 不幸的是，安全有效的治疗干预措施或疫苗是不可用的许多病毒， 全球性的病原体。针对这些病原体的中和抗体（nAb）的鉴定 将能够进行被动血清治疗以及疫苗抗原（Ag）鉴定。然而，确定 由病毒感染引起的nAb的功能库面临两个令人生畏的挑战。首先，几个 基于免疫方法学使得抗体（Ab）发现成为可能。这些方法可靠地支持 产生多克隆血清，并与杂交瘤技术，单克隆抗体的可再生来源。 然而，多克隆抗体是不可再生的，杂交瘤的产生是昂贵的，耗时的，并且结果是不稳定的。 在Ab多样性的实质性损失中（即，有偏见的）。直接筛选大量原代B细胞是 是nAb发现的理想选择。然而，原代浆母细胞培养是具有挑战性的，并且Ab-1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不切实际。二、可用 用于测试Ab功能的测定是低通量的、昂贵的，并且需要大量的人力投入 和资源来执行。在这里，提出了一种可以解决所有这些挑战的创新方法。 由酵母细胞分泌的杂合性抗体，可以容易地培养，并且不干扰病毒感染， 宿主细胞构成了一种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），一种新型的液滴微流控平台，结合了高通量微流控和酵母抗体 分泌文库技术，快速鉴定抗病毒的nAb，显著提高通量 以及与传统系统相比相应的成本降低。重要的是，因为生物学 在酵母中表达的Ab的功能（病毒中和）直接在系统中测试，而不是简单地 通过Ag-Ab结合相互作用，预计FAST将返回高质量、有生物学意义的命中。到目前为止， 令人兴奋的概念验证数据显示，FAST系统可用于识别中和抗体， 已收集鼠肝炎病毒（MHV）感染。为了检验FAST将提供 一种快速、低成本、无偏见的鉴定中和病毒感染的抗体的方法，该提议将：AIM 1. 通过使用模型系统针对MHV执行模拟筛选来优化FAST; AIM 2。为了测试工作 假设来自MHV感染小鼠的Ab文库含有有效的MHV中和分子。如果 成功后，这项工作将建立第一个高通量系统进行功能性病毒中和 单细胞（数字）分辨率的生物测定，允许识别针对全球病毒病原体的nAb 后果

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.