COVID-19: Elucidating monoclonal and polyclonal seroantibody responses to the COVID-19 viral envelope

背景/依据：随着2019年冠状病毒病（COVID-19）疫情在美国各地的蔓延， 在美国和全世界，没有经过证实的治疗方法，关于免疫力的信息也很少。 这个病毒。在这一流行病的现阶段，所有有希望的预防和治疗战略都必须 探讨了本建议将集中在多克隆和单克隆抗体反应的严重急性 呼吸综合征冠状病毒2型（SARS-CoV-2）包膜。 目标：该计划的总体目标是获得全面了解 对SARS-CoV-2包膜E、M和S蛋白的多克隆和单克隆应答。的具体目标 本研究的主要内容是：1）解卷积SARS-CoV-2病毒包膜的多克隆抗体反应; 2)分离针对刺突蛋白（S）的各种表位的中和和非中和单克隆抗体， SARS-CoV-2的囊膜蛋白（E）和膜糖蛋白（M）; 3）绘制相应的 单克隆抗体的表位。 方法：我们将从60例住院和门诊的急性和恢复期患者中获得配对样本， COVID-19（30人已经从弗吉尼亚州马里兰州卫生保健系统和弗吉尼亚大学入学） 马里兰州医疗系统）。我们将通过结合、活病毒和假病毒中和来筛选样品。在这 根据这项提议，我们还可以使用该园区的BSL 3设施，这些设施可以使用活病毒进行中和试验 作者：世界冠状病毒专家之一（马特·弗里曼博士）。捐助者将根据这两个因素进行排名 中和的效力和广度，以及结合力。住院病人和门诊病人的前三名捐赠者 将选择组进行进一步研究（最高抗RBD中和和呼吸，最高抗受体结合结构域 （RBD）耗尽的血浆中和和呼吸，以及非中和血浆的最高峰结合滴度）。 在这六个个体中，抗包膜抗体将被亲和纯化;并且使用游离的抗包膜抗体进行分级。 流动电泳这种技术可以根据电荷分离抗体，这将导致分离 也是基于靶向表位。将通过结合和中和对单个组分进行检测;以及 将确定靶向每个表位的抗体的特征性生物化学和功能特征。 感兴趣的部分（取决于靶向中和或结合，每个供体不同）将被发送至 质谱分析法来B细胞文库也将由恢复期IgG和伊加记忆B细胞库制备; 并将使用三种互补技术对其进行询问，包括急性期浆母细胞 库分析、减法分析和抗原诱食以鉴定潜在的抗体。 最后，将使用质谱法对候选抗体进行排序。40种单克隆抗体（mAb） 通过中和效价、宽度和等电点进行筛选， 和结合抗体（与靶向的级分的相应谱匹配）向前移动 用于通过X射线晶体学进行精细表位分析。 影响：如果成功，该项目将产生对中和/非中和的实质性理解 新冠病毒病中的抗体和表位，提供可用于动物/人类测试的mAb。 这项研究与退伍军人的健康直接相关，因为任何分离的单克隆抗体都可以 可能用于治疗和/或预防COVID-19。

Background/Rationale: As the Coronavirus Disease 2019 (COVID-19) epidemic expands across the United States and the world, there are no proven therapies and little information is known regarding on immunity to this virus. At this stage of the pandemic, all prevention and treatment strategies that show promise must be explored. This proposal will focus on the polyclonal and monoclonal antibody responses to the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) envelope. Objectives: The overarching goal of this program is to obtain a comprehensive understanding of the polyclonal and monoclonal response to the SARS-CoV-2 envelope E, M, and S proteins. The specific aims of this proposal are: 1) Deconvolute the polyclonal antibody response against the viral envelope of SARS-CoV-2; 2) Isolate neutralizing and non-neutralizing monoclonal antibodies against various epitopes of spike protein (S), envelope protein (E), and membrane glycoprotein (M) of SARS-CoV-2; and 3) Map the corresponding epitope(s) of the monoclonal antibodies. Methods: We will obtain paired, acute and convalescent, samples from 60 inpatients and outpatients with COVID-19 (30 have already been enrolled from the VA Maryland Health Care System and the University of Maryland Medical System). We will screen samples by binding, live and pseudovirus neutralization. In this proposal, we also will have access to BSL3 facilities on this campus that run neutralization assay with live virus by one of the world experts in coronaviruses (Matt Frieman, PhD). Donors will be ranked on the basis of both neutralization potency and breadth, and binding. The top three donors in each of the inpatient and outpatient groups will be chosen for further study (top anti-RBD neutralization and breath, top anti- receptor binding domain (RBD)-depleted plasma neutralization and breath, and top spike binding titers with non-neutralizing plasma). In these six individuals, the anti-envelope antibody will be affinity purified; and fractionated using free- flow-electrophoresis. This technique can separate antibodies based on charge, which will lead to separation based on targeted epitopes as well. Individual fractions will be tested by binding and neutralization; and characteristic biochemical and functional signatures of antibodies targeting each epitope will be ascertained. Fractions of interest (different for each donor depending if neutralization or binding is targeted) will be sent for mass spectrometry. B cell libraries will also be made from the convalescent IgG and IgA memory B cell pools; and they will be interrogated using three complementary techniques including - acute phase plasmablast repertoire analysis, subtraction analysis, and antigen baiting to identify potential antibodies. Finally, mass spectrometry will be used to rank antibody candidates. 40 monoclonal antibodies (mAbs) will be made and again be screened by neutralization potency, breadth, and isoelectric point, with top neutralizing and binding antibodies (matching the respective profiles of the fractions that were targeted) to be moved forward for fine epitope analysis by X-ray crystallography. Impact: If successful, this project will yield a substantial understanding of neutralizing/non-neutralizing antibodies and epitopes in COVID-19 disease, providing mAbs that can be moved into animal/human testing. This research has direct relevance to the health of Veterans as any monoclonal antibodies isolated can potentially be used for treatment and/or prevention of COVID-19.