Development of a High-Throughput Microfluidics-Enabled Functional Assay for Rapidly Identifying Neutralizing Antibodies

摘要 病毒致病每年导致1亿人死亡。然而，广谱中和抗体(Abs)有利于- 由幸运的宿主体内的B淋巴细胞诱导，通常可以消除最致命的病毒的威胁。识别 能够通过免疫诱导这种中和抗体(NaB)的抗原表位充其量是持久的和 充满了技术挑战，如果可能的话。我们正在开发一种新型的微流控芯片实验室技术- 从根本上加快从功能上分析由 一个人类B细胞。Precient(使用集成的MIMS快速评估抗体成功的平台) Croflidics Enabling Technology)是一个高通量、单细胞分辨率的平台，可以测量神经- 单个B淋巴细胞产生的抗体在液滴中直接功能测定的转化能力 微流控格式。因此，它提供了无偏、单细胞分辨率、高通量、近乎完整的肛门- 分析整个B细胞谱系，以确定中和病毒感染的B细胞。 由于流感有时每年导致50万人死亡，而我们有一系列工具和理由-- 绅士已经为这种病理系统开发，我们选择它作为病毒模型，在其中测试我们的中央 有先见之明的假设将为发现中和抗体(NAB)提供一条快速且低成本的途径。我们 将采取三方方法，以实现以下目标：1)以更高的可靠性优化设备的性能- 能力、吞吐量和效率；2)定量评估先知者识别杂交瘤的能力 从日益严格的混合人群中制定针对H1N1流感的nabs，以及3)快速识别- 从EBV永生化的人外周血B细胞中提取抗H1N1和H3N2型流感的nabs。 噬菌体和其他通常用于NAB发现的展示系统具有固有的偏见和蛋白质产物。 翻译障碍是由于非哺乳动物的翻译后修饰，因此直接利用B细胞是理想的。 液滴微流控系统，其中微升规模的油包水乳液液滴作为独立的生物 反应堆，可以以前所未有的速度和精度高效地操纵细胞。液滴微流控系统 为了筛选杂交瘤产生的抑制特定生化反应的抗体，已经描述了这些抗体。 然而，将抗体筛选和病毒中和生物检测相结合的系统尚未实现。 主要的创新是开发和使用了第一个高通量的功能性系统 人类抗感染性抗体的发现，是疫苗学、免疫学、免疫学等领域真正的垂直飞跃。 治疗设计和表位发现。有了先见之明，免疫学家将能够迅速识别 恢复期患者的血液产生了广泛的救命作用，这是大海捞针。 中和Abs。最终，独特的免疫球蛋白重链和轻链DNA重排将是等同的- 从预分选的细胞中分离出来，用于生产单抗或结构疫苗表位工程。 1

ABSTRACT Viral pathogenesis kills 100 million people each year. However, broadly neutralizing antibodies (Abs) pro- duced by B lymphocytes in fortunate hosts routinely eliminates the threat of the most lethal viruses. Identifying the antigenic epitopes that can induce such neutralizing Abs (nAbs) via immunization is at best protracted and fraught with technical challenges, if possible at all. We are developing a novel, microfluidic, lab-on-a-chip tech- nology to radically speed the ability to functionally assay the neutralization capability of clonal Ab produced by one human B cell. PRESCIENT (Platform for the Rapid Evaluation of antibody SucCess using Integrated mi- crofluidics ENabled Technology) is a high-throughput, single-cell resolution platform that can measure the neu- tralization capability of the Abs produced by single B lymphocytes through a direct functional assay in a droplet microfluidics format. Thus, it provides an unbiased, single-cell resolution, high-throughput, near-complete anal- ysis of the entire B cell repertoire to identify B cells that neutralize viral infection. As influenza sometimes causes one half million deaths per year and we have a battery of tools and rea- gents already developed for this pathosystem, we have chosen it as the viral model in which to test our central hypothesis that PRESCIENT will deliver a fast and low cost route for discovering neutralizing Abs (nAbs). We will take a tripartite approach with these aims: 1) to optimize the performance of the device with greater reliabil- ity, throughput and efficiency, 2) to quantitatively assess PRESCIENT's ability to recognize hybridomas that make nAbs against H1N1 influenza from progressively more rigorous mixed populations, and 3) to rapidly iden- tify nAbs against H1N1 and H3N2 influenza from EBV-immortalized human peripheral blood B cells. Phage and other display systems commonly used for nAb discovery have inherent bias and protein produc- tion hurdles due to non-mammalian post-translational modifications, thus direct utilization of B cells is ideal. Droplet microfluidic systems, where pico-liter scale water-in-oil emulsion droplets function as independent bio- reactors, can efficiently manipulate cells with unprecedented speed and precision. Droplet microfluidic systems for screening Abs produced from hybridomas that inhibit specific biochemical reactions have been described. However, systems that integrate Ab screening and viral neutralization bioassays have not yet been achieved. The major innovation is the development and utilization of the first high-throughput system for the functional discovery of human nAbs against infectious agents, a truly vertical leap for the fields of vaccinology, immuno- therapeutic design and epitope discovery. With PRESCIENT, immunologists will be able to rapidly identify from a convalescent patient's blood draw the “needle in the haystack” paratope that generates life-saving broadly neutralizing Abs. Ultimately, the unique immunoglobulin heavy and light chain DNA rearrangements will be iso- lated from the PRESCIENT-sorted cell for monoclonal Ab production or structural vaccine epitope engineering. 1