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痛苦，每年有超过10亿人感染病毒，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。抗这些病原体的中和抗体的鉴定 将使被动血清疗法以及疫苗抗原(Ag)鉴定成为可能。然而，确定 由病毒感染引发的nabs的功能体系面临两个艰巨的挑战。首先，有几个 基于免疫的方法使抗体(Ab)能够被发现。这些方法可靠地支持 制备多克隆血清，并利用杂交瘤技术，获得可再生的单抗来源。 然而，多克隆抗体是不可再生的，杂交瘤的产生昂贵、耗时和结果 在很大程度上丧失了抗体多样性(即，有偏见)。直接筛选大量的原代B细胞 NAB发现的理想选择。然而，原代成浆细胞培养是具有挑战性的，而抗体-1的有限寿命。 在培养中产生原代成浆细胞使直接筛选这些细胞变得不切实际。第二，可用 用于测试抗体功能的检测方法吞吐量低、成本高，并且需要大量的人力 以及可供执行的资源。在这里，提出了一种创新的方法，可以应对所有这些挑战。 酵母细胞分泌异源抗体，易于培养，不干扰病毒感染 宿主细胞，构成了一个有吸引力的选择。这一建议将迅速发展(功能性抗体筛查 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，快速识别针对病毒的NAB，实现吞吐量的大幅增加 与传统系统相比，成本也相应降低。重要的是，因为生物 在酵母中表达的抗体(病毒中和)的功能是在系统中直接测试的，而不是简单的 Ag-Ab结合相互作用，FAST有望返回高质量的、具有生物学意义的命中结果。到目前为止， 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 已收集到小鼠肝炎病毒(MHV)感染。要检验FAST将交付的核心假设 这是一种快速、低成本、不偏不倚的识别中和病毒感染的抗体的方法，该提案将：目标1。 通过使用针对MHV的模型系统执行模拟屏幕来快速优化；目标2.测试工作 假设来自MHV感染小鼠的抗体文库包含有效的MHV中和分子。如果 这项工作的成功将建立第一个进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，允许识别针对全球 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.