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

由SARS-CoV-2冠状病毒感染引起的新冠肺炎目前是一场全球性的大流行。最多的 重要的问题是，为什么有些人产生快速保护性抗体(Abb)而表现为空或仅有 轻微的症状，而其他人则没有？第二个紧迫的问题是，标准的实验室检测是否可以 对最有可能形成快速、长期存活的B细胞的受试者进行分层，这些B细胞可以产生 感染后还是接种后的保护性抗体？基于对有效的抗体应答的理解 对于其他冠状病毒，简单的感染会促进对尖峰蛋白(S蛋白)的应答以及对 病毒核蛋白(VNP)。这项建议将集中在干扰素-β(干扰素-β)的作用，它可以增强B细胞 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细胞内源性干扰素-β使B细胞谱系的发展偏向于 识别内源性核糖核蛋白(RNPs)，我们建议形成免疫谱系 准备对vNPs做出回应的人。在SARS-CoV-2的情况下，VNP是一个正链RNA复合体，与 核蛋白一种核蛋白，与人类体内发育的天然蛋白质非常相似。对于2003年的SARS-CoV，VNP 在感染早期的多种体液中被检测到，并且已经证明抗NP的免疫球蛋白是长期存在的 在康复患者中升高。我们已经证明，I型干扰素促进长寿的血浆B细胞 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。一种类似的 对SARS-CoV-2 S蛋白产生保护性抗体应答的机制进行了探讨。在一次 在机制水平上，我们提出对S蛋白的高亲和力反应也需要增强单个 链RNA诱导的Toll样受体7信号转导与滤泡T细胞因子尤其是II型干扰素 将B细胞转化为长寿浆细胞(PC)。特异性靶点1将确定新冠肺炎中的B细胞 内源性干扰素-β升高的恢复期患者产生较强的抗-VNP和S蛋白反应。这 将用荧光标记抗原的策略来鉴定VNP和S蛋白特异的B细胞 并用流式细胞仪分析这些B细胞的表型。特定目标2将利用荧光和核苷酸 条形码编码vnp和S蛋白，使5‘10X基因组测序能够确定程序 与B细胞发育相关的I型和II型干扰素反应途径 腹肌。这些研究通过我们过去对B细胞抗体的发展和反应的研究而变得可行 恢复的受试者表现出高内源性干扰素-β，并产生抗VNP或S蛋白的抗体。克隆， 将进行VNP和S蛋白特异性B细胞产生的抗体的测序、生产和检测 利用5‘10X基因组和Twist方法将重链和轻链克隆到表达载体中 生物技术。来自伯明翰VAMC的一支杰出的调查团队已经组建完毕 包括约翰·芒茨博士和约翰·卡普斯博士，前者擅长B细胞开发，后者擅长于 病毒学。我们已经招募了在单细胞分析和免疫学方面具有专业知识的科学家来协助 项目。这些研究具有非常重要的意义，因为它们将导致更好地理解和分层 在感染后产生和没有产生有效的SARS-CoV-2病毒抗体反应的受试者以及 确定有效应对所需的潜在免疫环境。这些研究将是重要的 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。这些研究还将 立即应用于与抗病毒开发相关的潜在免疫调节治疗 反应，如羟氯喹的潜在机制，它可以调制TLR7信号和其他 进入细胞后病毒处理的各个方面。其他直接的应用将是指导开发 先天免疫反应和获得性免疫反应，以增强和实现长期的B细胞免疫。

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.