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

COVID-19：阐明对 COVID-19 病毒包膜的单克隆和多克隆血清抗体反应

• 批准号: 10513290
• 负责人: Mohammad Mohseni Sajadi
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-10-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10513290
• 关键词: 2019-nCoV; Acute; Affinity Chromatography; American; Animals; Antibodies; Antibody Response; Antigens; Award; B-Lymphocytes; Binding; Binding Sites; Biochemical; Biological Assay; COVID-19; COVID-19 pandemic; COVID-19 patient; COVID-19 prevention; Categories; Cells; Cessation of life; Characteristics; Charge; Codon Nucleotides; Coronavirus; Disease; Doctor of Philosophy; Electrophoresis; Enrollment; Epitope Mapping; Epitopes; Family; Fractionation; Goals; HIV; Hand; Health; Healthcare Systems; Human; Immunity; Immunoglobulin A; Immunoglobulin G; Individual; Infection; Inpatients; Isoelectric Point; Knowledge; Libraries; Maps; Maryland; Mass Spectrum Analysis; Medical; Membrane Glycoproteins; Memory B-Lymphocyte; Methods; Monoclonal Antibodies; Nucleoproteins; Outpatients; Patients; Persons; Phase; Plasma; Plasmablast; Plasmids; Prevention strategy; Production; Proteins; Protocols documentation; Research; Running; SARS-CoV-2 antibody; SARS-CoV-2 infection; SARS-CoV-2 spike protein; Sampling; System; Techniques; Testing; United States; Universities; Vaccines; Veterans; Viral; Virus; X-Ray Crystallography; biosafety level 3 facility; convalescent plasma; env Gene Products; experience; experimental study; interest; multiple myeloma M Protein; neutralizing antibody; novel therapeutics; novel vaccines; pandemic disease; participant enrollment; polyclonal antibody; programs; receptor binding; response; treatment strategy

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.

背景/理由：随着2019年冠状病毒病（Covid-19）的流行，整个联合的流行 各州和世界，关于免疫的疗法没有验证的疗法，很少有信息 该病毒。在大流行的这个阶段，所有预防和待遇策略都必须是 探索。该建议将重点放在对严重急性的多克隆和单克隆抗体的反应上 呼吸综合征冠状病毒2（SARS-COV-2）包膜。 目标：该计划的总体目标是获得对 对SARS-COV-2包膜E，M和S蛋白的多克隆和单克隆反应。具体目的 该提议是：1）反对SARS-COV-2病毒包膜的多克隆抗体反应； 2）孤立的中和和非中和单克隆抗体针对峰值蛋白的各种表位 SARS-COV-2的包膜蛋白（E）和膜糖蛋白（M）； 3）映射相应的 单克隆抗体的表位。 方法：我们将获得配对，急性和康复的样品，来自60个住院患者和门诊病人 COVID-19（30个已经从VA马里兰州医疗保健系统和大学注册 马里兰州医疗系统）。我们将通过结合，活病毒中和筛选样品。在这个 提案，我们还可以在该校园内使用使用现场病毒进行中和测定的BSL3设施 由冠状病毒（Matt Frieman，PhD）的世界专家之一。捐助者将在两者的基础上排名 中和效力和广度和结合。每个住院和门诊的前三名捐助者 将选择组进行进一步研究（顶部抗RBD中和和呼吸，顶部抗受体结合结构域 （RBD）缺失的血浆中和和呼吸，以及具有非中和血浆的顶部尖峰结合滴度）。 在这六个人中，抗Envelope抗体将具有亲和力；并使用自由 - 流动流体。该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。单个分数将通过结合和中和测试；和 将确定针对每个表位的抗体的特征性生化和功能特征。 感兴趣的分数（每个捐赠者不同，具体取决于中和或结合的目标）将被发送 质谱法。 B细胞库也将由康复IgG和IgA记忆B细胞池制成。 它们将使用三种互补技术（包括 - 急性相位plasmablast）进行审问 曲目分析，减法分析和抗原诱饵，以鉴定潜在的抗体。 最后，质谱法将用于对抗体候选。 40个单克隆抗体（mAb） 将制作并再次通过中和效率，宽度和等电点进行筛选，顶部中和 和结合抗体（与目标分数的各个分布匹配）要向前移动 为了通过X射线晶体学分析精细的表位。 影响：如果成功，该项目将对中和/非中和化产生深刻的理解 COVID-19疾病中的抗体和表位，提供可以转移到动物/人类测试中的mAb。 这项研究与退伍军人的健康直接相关，因为任何单克隆抗体孤立的都可以 可能用于治疗和/或预防Covid-19。