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

开发高通量微流控功能测定法以快速识别中和抗体

• 批准号: 10640278
• 负责人: PAUL DE FIGUEIREDO
• 金额: $ 13.15万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-07 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10640278
• 关键词: Accounting; Address; Advanced Development; Affect; Antibodies; Antibody Binding Sites; Antibody Repertoire; Area; B cell repertoire; B-Lymphocytes; Bacteriophages; Biochemical Reaction; Biological Assay; Bioreactors; Blood; Cell Separation; Cells; Cessation of life; Child; Communicable Diseases; Consumption; Data; Dengue Virus; Development; Devices; Disease Outbreaks; Elderly; Emulsions; Engineering; Environment; Epitopes; Evaluation; Flu virus; Gene Rearrangement; Genetic Recombination; HIV; Human; Human Herpesvirus 4; Hybridomas; Immunity; Immunization; Immunoglobulins; Immunologic Deficiency Syndromes; Immunologist; Immunology; Immunotherapeutic agent; Individual; Infection; Infectious Agent; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza Therapeutic; Laboratories; Libraries; Licensure; Life; Light; Measures; Membrane Proteins; Microfluidic Microchips; Microfluidics; Modeling; National Institute of Allergy and Infectious Disease; Needles; Oils; Outcome; Patients; Performance; Persons; Point Mutation; Population; Post-Translational Protein Processing; Production; Proteins; Qualifying; Reagent; Research; Research Support; Resistance; Resolution; Respiratory System; Route; SARS coronavirus; Serotyping; Speed; Strategic Planning; Syndrome; System; Technology; Testing; Texas; Therapeutic Intervention; Therapeutic antibodies; Time; United States National Institutes of Health; Universities; Vaccines; Variant; Viral; Viral Antigens; Viral Pathogenesis; Virus; Virus Diseases; Water; Work; Zika Virus; clinical application; cost; cross immunity; design; experimental study; flu; human coronavirus; human disease; improved; influenza infection; influenza virus strain; innovation; monoclonal antibody production; multidisciplinary; nanofabrication; neutralizing antibody; novel; pandemic influenza; pathogen; pathogenic virus; peripheral blood; rapid test; respiratory; response; screening; success; therapeutic vaccine; tool; vaccinology; virology

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

抽象的 病毒发病机制每年导致 1 亿人死亡，然而，广泛中和抗体 (Ab) 亲-。 幸运宿主体内的 B 淋巴细胞诱导的病毒通常可以消除最致命病毒的威胁。 通过免疫接种可诱导此类中和抗体 (nAb) 的抗原表位充其量是长期存在的，并且 如果可能的话，我们正在开发一种新颖的微流体芯片实验室技术，充满了技术挑战。 技术从根本上加快了功能测定克隆抗体中和能力的能力 一个人类 B 细胞（使用集成 mi- 快速评估抗体成功的平台） Crofluidics（ENabled 技术）是一种高通量、单细胞分辨率平台，可以测量神经 通过液滴中的直接功能测定对单个 B 淋巴细胞产生的抗体进行分析的能力 因此，它提供了无偏差、单细胞分辨率、高通量、近乎完整的分析。 分析整个 B 细胞库，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，我们拥有一系列工具和应对措施 男性已经针对这种病理系统开发出来，我们选择它作为病毒模型来测试我们的中枢 假设 PRESCIENT 将为发现中和抗体（nAb）提供一种快速且低成本的途径。 将采取三方方法来实现以下目标：1）以更高的可靠性优化设备的性能 性、通量和效率，2) 定量评估 PRESCIENT 识别杂交瘤的能力 从日益严格的混合人群中制造针对 H1N1 流感的 nAb，以及 3) 快速鉴定 从 EBV 永生化人外周血 B 细胞中提取针对 H1N1 和 H3N2 流感的 nAb。 噬菌体和其他常用于 nAb 发现的展示系统具有固有的偏差和蛋白质生产 由于非哺乳动物翻译后修饰造成的障碍，因此直接利用 B 细胞是理想的。 液滴微流体系统，其中皮升规模的油包水乳液液滴充当独立的生物 反应器，可以以前所未有的速度和精度有效地操纵细胞。 已经描述了用于筛选由杂交瘤产生的抑制特定生化反应的抗体的方法。 然而，整合抗体筛选和病毒中和生物测定的系统尚未实现。 主要创新是第一个功能性高通量系统的开发和利用 发现针对传染源的人类 nAb，这是疫苗学、免疫学领域真正的垂直飞跃 借助 PRESCIENT，免疫学家将能够快速识别治疗设计和表位。 康复期患者的血液抽取了“大海捞针”的互补位，可以广泛挽救生命 最终，独特的免疫球蛋白重链和轻链 DNA 重排将是 iso- 从 PRESCIENT 分选的细胞中提取，用于单克隆抗体生产或结构疫苗表位工程。 1