B-cell innate and adaptive protective immunity to SARS-CoV-2

B 细胞对 SARS-CoV-2 的先天性和适应性保护性免疫

• 批准号: 10778521
• 负责人: John D Mountz
• 金额: --
• 依托单位: BIRMINGHAM 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: 2021-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10778521
• 关键词: 2019-nCoV; Adjuvant; Affinity; Alabama; Antibodies; Antibody Affinity; Antibody Response; Antigens; Antiviral Response; Autoimmune; Autoimmune Diseases; B cell repertoire; B-Cell Development; B-Lymphocyte Subsets; B-Lymphocytes; B-cell receptor repertoire sequencing; Bar Codes; Biological Assay; Biotechnology; Body Fluids; CD86 gene; COVID-19; COVID-19 outbreak; COVID-19 surveillance; Cells; Cloning; Complement 3d Receptors; Complex; Core Facility; Coronavirus; Critical Care; Data Collection; Dendritic Cells; Development; Disease; Endosomes; Event; Exhibits; Feasibility Studies; Flow Cytometry; Future; Genes; Genomics; Helper-Inducer T-Lymphocyte; Herd Immunity; Human; Hydroxychloroquine; ISG15 gene; ITGAX gene; Immune; Immune response; Immunity; Immunoglobulin D; Immunoglobulin G; Immunology; Individual; Infection; Innate Immune Response; Interferon Type I; Interferon Type II; Interferon alpha; Interferon-beta; Interferons; Label; Laboratories; Light; Mature B-Lymphocyte; Mediating; Microbiology; Mus; Nuclear Protein; Nucleoproteins; Nucleotides; Pathogenesis; Pathway interactions; Patients; Peripheral; Phenotype; Plasma; Plasma Cells; Probability; Production; Proteins; RNA; Recurrence; Research Personnel; Research Project Grants; Ribonucleoproteins; Role; SARS coronavirus; SARS-CoV-2 antibody; SARS-CoV-2 antiviral; SARS-CoV-2 immunity; SARS-CoV-2 infection; SARS-CoV-2 spike protein; Scientist; Serology; Signal Transduction; Social Distance; Specimen Handling; Symptoms; Systemic Lupus Erythematosus; T-Lymphocyte; TLR7 gene; Technology; Testing; Vaccination; Vaccine Design; Viral; Viral Genes; Virus; adaptive immune response; antiviral drug development; contagion; cytokine; expression vector; immunomodulatory therapies; interleukin-21; mid-career faculty; monocyte; neoantigens; pandemic disease; plasma cell development; prevent; programs; recruit; response; single cell analysis; single-cell RNA sequencing; transcriptomics; virology

The COVID-19 caused by the SARS-CoV-2 coronavirus infection is currently a global pandemic. The most important question is why some individuals produce rapid protective antibodies (Abs) and exhibit null or only mild symptoms, whereas others do not? A second urgent question is if a standard laboratory test can be developed to stratify subjects who would be most likely to develop rapid long-lived B cells that could produce protective Abs after infection or after vaccination? Based on the understanding of efficient Ab responses to other coronavirus, an uncomplicated infection would promote a response to the spike (S-protein) as well as to the viral nucleoprotein (vNP). This proposal will focus on the role of interferon-β (IFN-β) that enhances the B cell Ab response. Our previous studies in autoimmune systemic lupus erythematosus (SLE) patients have shown that high levels of B cell endogenous IFN-β enables development of a B cell repertoire skewed toward recognition of an endogenous ribonuclear protein (RNPs) which we propose then form the immune repertoire that is poised to respond to vNPs. In the case of SARS-CoV-2, the vNP is a positive strand RNA complex with a nuclear protein very similar to the natural repertoire that develops in humans. For the 2003 SARS-CoV, vNP was detected in multiple body fluids during early infection and IgG anti-NP has been shown to be long-lived and elevated in recovered patients. We have shown that type I IFN promotes long-lived plasma B cell development including T-cell mediated development of high affinity antibody to a neo-antigen. A similar mechanism is proposed for development of protective Ab response to the S-protein of SARS-CoV-2. At a mechanistic level, we propose that the high affinity response to S-protein also requires enhanced single stranded RNA-induced toll-like receptor 7 (TLR7) signaling and follicular T-cell cytokines especially type II IFN to propel B cell into long-lived plasma cells (PCs). Specific Aim 1 will determine if B cells from COVID-19 convalescent subjects with high endogenous IFN-β produce a strong anti-vNP and S-protein response. This will be investigated using fluorescent labeling of antigen strategy to identify vNP and S-protein-specific B cells and to phenotype these B cells by flow cytometry analysis. Specific Aim 2 will utilize fluorescent and nucleotide barcoded vNP and S-protein to enable analysis by 5' 10X Genomics sequencing to determine the program of type I and type II IFN response pathways that are associated with development of B cells producing protective Abs. These studies are made feasible by our past studies of B cell Ab development and responses from recovered subjects that exhibit high endogenous IFN-β and produce antibodies to vNP or S-proteins. Cloning, sequencing, production and testing of the Ab produced by the vNP and S-protein specific B cells will be carried out using 5' 10X Genomics and cloning of heavy and light chain regions into an expression vector by TWIST Biotechnologies. An outstanding team of investigators from the Birmingham VAMC has been assembled including Dr. John Mountz, with expertise in B cell development and Dr. John Kappes, with expertise in virology. We have recruited scientists with expertise in single-cell analysis and immunology to assist with the project. The studies are highly significant since they will lead to a better understanding and stratifications of subjects who did and did not generate an efficient Ab response of the SARS-CoV-2 virus after infection and to determine the underlying immune landscape required for an efficient response. The studies will be important in the design of vaccines to understand the combined role of the vNP and S-protein of SARS-CoV-2, as well as adjuvant that can promote an efficient interferons and TLR7-mediated B cell response. The studies will also have immediate applications to potential immunomodulatory therapy related to development of the anti-viral response such as a potential mechanism of hydroxychloroquine which can modulate TLR7 signaling and other aspects of viral processing upon entry into cells. Other immediate applications would be to guide development of both innate and adaptive immune responses to boost and enable a long-term B cell immunity.

由 SARS-CoV-2 冠状病毒感染引起的 COVID-19 目前是全球大流行。最 重要的问题是为什么有些人会产生快速保护性抗体（Abs）并且表现出无效或仅表现出 症状轻微，而其他人则没有？第二个紧迫问题是标准实验室测试是否可以 开发用于对最有可能发育出快速长寿 B 细胞的受试者进行分层，这些 B 细胞可以产生 感染后或疫苗接种后有保护性抗体吗？基于对有效抗体反应的理解 与其他冠状病毒一样，简单的感染会促进对尖峰（S-蛋白）以及对 病毒核蛋白（vNP）。该提案将重点关注干扰素-β (IFN-β) 增强 B 的作用。 细胞抗体反应。我们之前对自身免疫性系统性红斑狼疮 (SLE) 患者的研究 研究表明，高水平的 B 细胞内源性 IFN-β 能够促进 B 细胞库的发育 识别内源性核糖核蛋白（RNP），我们建议将其形成免疫库 准备响应 vNP。就 SARS-CoV-2 而言，vNP 是一种正链 RNA 复合物， 一种与人类自然发育的核蛋白非常相似的核蛋白。对于 2003 年 SARS-CoV，vNP 在早期感染期间在多种体液中检测到 IgG 抗 NP，已证明其寿命较长 并在康复患者中升高。我们已经证明 I 型 IFN 可以促进血浆 B 细胞的长寿 开发包括T细胞介导的针对新抗原的高亲和力抗体的开发。类似的 提出了针对 SARS-CoV-2 S 蛋白产生保护性抗体反应的机制。在一个 机制水平上，我们提出对S蛋白的高亲和力反应也需要增强的单 链状 RNA 诱导的 Toll 样受体 7 (TLR7) 信号传导和滤泡 T 细胞细胞因子，尤其是 II 型 IFN 推动 B 细胞转化为长寿命浆细胞 (PC)。具体目标 1 将确定 B 细胞是否来自 COVID-19 具有高内源性 IFN-β 的恢复期受试者会产生强烈的抗 vNP 和 S 蛋白反应。这 将使用荧光标记抗原策略来识别 vNP 和 S 蛋白特异性 B 细胞 并通过流式细胞仪分析对这些 B 细胞进行表型分析。具体目标 2 将利用荧光和核苷酸 带条形码的 vNP 和 S 蛋白，可通过 5' 10X 基因组测序进行分析，以确定 I 型和 II 型 IFN 应答途径与产生保护性 B 细胞的发育相关 绝对值。我们过去对 B 细胞抗体发育和反应的研究使这些研究变得可行 恢复的受试者表现出高内源性 IFN-β 并产生 vNP 或 S 蛋白抗体。克隆， 将进行 vNP 和 S 蛋白特异性 B 细胞产生的抗体的测序、生产和测试 使用 5' 10X Genomics 并通过 TWIST 将重链和轻链区域克隆到表达载体中 生物技术。来自伯明翰 VAMC 的优秀调查员团队已经组建 包括拥有 B 细胞开发专业知识的 John Mountz 博士和拥有 B 细胞开发专业知识的 John Kappes 博士 病毒学。我们招募了具有单细胞分析和免疫学专业知识的科学家来协助 项目。这些研究非常重要，因为它们将导致更好的理解和分层 感染后对 SARS-CoV-2 病毒产生或未产生有效抗体反应的受试者 确定有效反应所需的潜在免疫环境。研究将很重要 在疫苗设计中了解 SARS-CoV-2 的 vNP 和 S 蛋白的联合作用，以及 佐剂，可以促进有效的干扰素和 TLR7 介导的 B 细胞反应。研究还将 可以直接应用于与抗病毒药物开发相关的潜在免疫调节疗法 反应，例如羟氯喹的潜在机制，它可以调节 TLR7 信号传导和其他 病毒进入细胞后的加工过程。其他直接应用是指导开发 先天性和适应性免疫反应，以增强并实现长期 B 细胞免疫。