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

背景/理由：随着冠状病毒病2019(新冠肺炎)疫情在美国蔓延 在美国和世界上，还没有经过验证的治疗方法，关于免疫的信息也很少。 这种病毒。在大流行的这个阶段，所有显示出希望的预防和治疗战略都必须 探索过了。这项建议将集中在多克隆和单克隆抗体对严重急性冠状病毒的反应。 呼吸综合征冠状病毒-2(SARS-CoV-2)外膜。 目标：本课程的主要目标是全面了解 SARS-CoV-2囊膜蛋白E、M和S的多克隆和单克隆反应。的具体目标 这一建议是：1)解开针对SARS-CoV-2病毒包膜的多克隆抗体反应； 2)分离抗S蛋白各表位的中和和非中和单抗(S)， SARS-CoV-2的包膜蛋白(E)和膜糖蛋白(M)；以及3)映射相应的 单抗的表位(S)。 方法：收集60例住院和门诊患者的急性期和恢复期配对标本。 新冠肺炎(已有30人从退伍军人事务部马里兰州医疗保健系统和加州大学 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本。在这 提议，我们还将可以使用该校园内运行活病毒中和测试的BSL3设施 世界冠状病毒专家之一(马特·弗里曼博士)。捐赠者将根据这两个因素进行排名 中和效力和广度，并具有约束力。住院患者和门诊患者的前三位捐赠者 将选择组进行进一步研究(顶级抗RBD中和呼吸，顶级抗受体结合结构域 (RBD)耗尽的血浆中和和呼吸，以及与非中和血浆的顶峰结合滴度)。 在这六个人中，抗包膜抗体将被亲和纯化；并使用游离- 流动电泳法。这项技术可以根据电荷分离抗体，这将导致分离 也基于目标表位。个别组份将通过结合和中和进行测试；以及 将确定针对每个表位的抗体的特征生化和功能特征。 感兴趣的部分(每个供体不同，取决于是否针对中和或结合)将被发送用于 质谱学。还将从恢复期的免疫球蛋白和免疫球蛋白A记忆B细胞库建立B细胞库； 他们将使用三种互补的技术进行讯问，包括-急性时相浆母细胞 谱系分析、消减分析和抗原诱饵以识别潜在抗体。 最后，将使用质谱学对候选抗体进行排名。40株单抗(MAbbs) 将被制成，并再次通过中和效力、宽度和等电点进行筛选，具有最高的中和力 以及要向前移动的结合抗体(与目标组分的各自轮廓匹配) 用于X射线结晶学的精细表位分析。 影响：如果成功，该项目将对中和/非中和有实质性的理解 新冠肺炎病中的抗体和表位，提供了可以转移到动物/人体试验中的单抗。 这项研究与退伍军人的健康直接相关，因为任何分离的单抗都可以 有可能用于新冠肺炎的治疗和/或预防。

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.