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

由SARS-CoV-2冠状病毒感染引起的COVID-19目前是全球大流行。最 一个重要问题是，为什么有些个体产生快速保护性抗体（Abs）， 轻微的症状，而其他人没有？第二个紧迫问题是是否可以进行标准实验室测试 开发用于对最有可能发展快速长寿B细胞的受试者进行分层， 感染后或接种疫苗后的保护性抗体？基于对有效抗体应答的理解， 与其他冠状病毒一样，简单的感染会促进对刺突（S蛋白）的反应， 病毒核蛋白（vNP）。该建议将重点放在干扰素-β（IFN-β）的作用，增强B 细胞抗体应答。我们以前在自身免疫性系统性红斑狼疮（SLE）患者中的研究， 显示高水平的B细胞内源性IFN-β使得B细胞库的发展偏向于 我们提出的内源性核糖核蛋白（RNP）的识别然后形成免疫库 已准备好响应vNP。在SARS-CoV-2的情况下，vNP是正链RNA复合物， 一种核蛋白质，与人类体内发育的天然功能非常相似。对于2003年SARS-CoV，vNP 在早期感染期间在多种体液中检测到，并且IgG抗NP已被证明是长期存在的 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细胞介导的对新抗原的高亲和力抗体的开发。类似的 提出了SARS-CoV-2的S蛋白的保护性抗体应答的发展机制。在 机制水平，我们提出，高亲和力的反应，S-蛋白也需要增强的单一 单链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 以将B细胞推进为长寿的浆细胞（PC）。具体目标1将确定COVID-19的B细胞是否 具有高内源性IFN-β的恢复期受试者产生强的抗vNP和S蛋白应答。这 将使用荧光标记抗原策略进行研究，以鉴定vNP和S蛋白特异性B细胞 并通过流式细胞术分析对这些B细胞进行表型。Specific Aim 2将利用荧光和核苷酸 条形码化的vNP和S蛋白，以使得能够通过5 '10 X Genomics测序进行分析，以确定VNP和S蛋白的程序。 I型和II型IFN应答途径与产生保护性IFN的B细胞的发育有关， ABS.这些研究是可行的，我们过去的研究B细胞抗体的发展和反应， 恢复的受试者表现出高内源性IFN-β并产生针对vNP或S蛋白的抗体。克隆、 将对vNP和S蛋白特异性B细胞产生的Ab进行测序、生产和检测 使用5'10X Genomics并通过TWIST将重链和轻链区克隆到表达载体中 生物技术。伯明翰VAMC的一个优秀的调查小组已经成立 包括约翰·蒙茨博士，他在B细胞发育方面有专长，约翰·卡普斯博士，他在 病毒学我们已经招募了具有单细胞分析和免疫学专业知识的科学家， 项目这些研究非常重要，因为它们将导致更好地理解和分层， 受试者在感染后对SARS-CoV-2病毒产生了有效的抗体应答， 确定有效反应所需的潜在免疫景观。这些研究很重要 在疫苗设计中，了解SARS-CoV-2的vNP和S蛋白的联合作用，以及 可促进有效的干扰素和TLR 7介导的B细胞应答的佐剂。这些研究还将 直接应用于与抗病毒药物的开发相关的潜在免疫调节治疗 反应，如羟氯喹的潜在机制，它可以调节TLR7信号和其他 病毒进入细胞后的处理方面。其他直接的应用将是指导发展 先天性和适应性免疫反应，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.