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 病毒发病机理每年杀死1亿人。但是，广泛中和抗体（ABS）促抗体 幸运宿主的B淋巴细胞通常消除了最致命的病毒的威胁。识别 可以通过免疫诱导这种中和ABS（NAB）的抗原表位最多是持久的，并且 如果可能的话，面对技术挑战。我们正在开发一种新颖的，微流体，实验室芯片技术 - NOOLOGAL从根本上加快了功能上主张克隆AB的神经化能力的能力 一个人类B细胞。先见者（使用综合MI-i-快速评估抗体成功的平台 启用Crofluidics的技术）是一个高通量的单细胞分辨率平台，可以测量neu- 单个B淋巴细胞通过液滴中的直接功能分析产生的ABS的Tralization能力 微流体格式。那就提供了公正的单细胞分辨率，高通量，几乎完整的肛门 整个B细胞库的YSIS识别中和病毒感染的B细胞。 由于影响力有时每年会造成一十万人死亡，我们有一系列工具和重新 已经为该悲伤系统开发的绅士，我们选择了它作为测试我们的中心的病毒模型 假设有先见之星将提供一条快速，低成本的途径，以发现中和ABS（NABS）。我们 将采用这些目的采用三方方法：1）以更大的可靠性优化设备的性能 - 它，吞吐量和效率，2）定量评估Prepient识别杂交瘤的能力 从逐渐严格的混合种群和3）到快速识别的H1N1影响力进行NABS。 从EBV侵蚀性人类外周血B细胞中针对H1N1和H3N2影响的NABS。 噬菌体和其他用于NAB发现的显示系统具有继承偏见和蛋白质的产生 - 由于非哺乳动物的翻译后修饰引起的障碍，因此直接利用B细胞是理想的。 液滴微流体系统，其中Pico-liter尺度水中水乳液液滴起独立的生物 - 反应堆可以以前所未有的速度和精度有效地操纵细胞。液滴微流体系统 为了筛选毒性瘤产生的ABS，已经描述了抑制特异性生化反应的ABS。 但是，尚未实现整合AB筛查和病毒神经化生物测定的系统。 主要创新是功能性的第一个高通量系统的开发和利用 发现人类NABS针对感染剂，这是疫苗学领域的真正垂直飞跃，免疫 - 热设计和表位发现。使用先见之明，免疫学家将能够从 康复患者的血液吸引了“干草堆中的针头”伞形，从而​​广泛产生寿命 中和腹肌。最终，独特的免疫球蛋白重链和轻型链DNA重排将是iso- 从先知分级的细胞中层压，用于单克隆AB的产生或结构蒸气表位工程。 1