Identification of neutralizing epitopes on SARS-CoV-2 spike for design of vaccines and small-molecule antivirals

鉴定 SARS-CoV-2 刺突上的中和表位，用于设计疫苗和小分子抗病毒药物

• 批准号: 10267406
• 负责人: Dennis R. Burton
• 金额: $ 63.56万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10267406
• 关键词: Animal Model; Animals; Antibody Response; Antigens; Biological Assay; Cells; Clinical Trials; Development; Exposure to; Future; Glycoproteins; Goals; HIV; HIV Infections; HIV Vaccine Trials Network; HIV vaccine; Human; Immunization; Immunoglobulin Somatic Hypermutation; Immunologics; Individual; Infection; Mucous Membrane; Phase I Clinical Trials; Pre-Clinical Model; Procedures; Regimen; Research Support; Sampling; Series; Site; Structure; Technology; Testing; Translating; Vaccines; Virion; Virus; design; experience; flexibility; in vivo; innovation; mouse model; neutralizing antibody; nonhuman primate; novel; operation; pre-clinical; product development; programs; protective efficacy; research clinical testing; response; success; vaccine development

Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses and are divided into Alphaand Beta-coronaviruses. CoVs infect mammals and birds and typically result in lower and/or upper respiratory tract disease. The spectrum of illness in humans caused by CoVs range from common colds to worldwide epidemics/pandemics, including severe acute respiratory syndrome (SARS-CoV) in 2003, human CoV-NL63 in 2004, human CoV-HKU1 in 2005, Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, and SARS-CoV-2 in 2019. There are no approved vaccines or antiviral drugs to combat CoV infections and a lack of tested and validated therapeutics represents a tremendous global concern with respect to the current SARSCoV-2 outbreak. At the end of December 2019, the World Health Organization became aware of an abnormally large cluster of pneumonia cases localized in the city of Wuhan, China. Within a span of only 3 months, over one million confirmed cases of COVID-19 have been diagnosed worldwide with almost 50,0000 resulting in death. The numbers in the US are now growing at an alarming rate (currently ~25% of the global total) as well as the number of deaths (currently ~10% of total). The severity of human CoV infections and high mortality rates were strikingly apparent in 2002 with the first SARS-CoV pandemic in Guangdong, China, as well as the MERS-CoV outbreak in 2012. Like SARS-CoV, the current SARS-CoV-2, which is 79% identical, also employs angiotensin converting enzyme II (ACE2) as the host receptor for cellular entry. The CoV surface-exposed spike (S) protein is responsible for the recognition and binding of ACE2 and represents a potential target for development of vaccines and antiviral therapeutics. Importantly, antibodies (Abs) isolated to date from COVID-19 patients appear to bind several regions on the spike protein that then represent ideal targets for small molecule discovery. The spike protein on the CoV surface is a glycosylated trimer and consists of two extracellular domains, S1 and S2. The majority of nAbs (neutralizing Abs) to CoVs characterized to date target the S1 domain that contains the receptor-binding domain (RBD) responsible for ACE2 binding. Notwithstanding, Abs with epitopes on the S2 domain, consisting of the stem fusion machinery, also have neutralizing potential in both cell-based and animal models of infection and are generally more broadly reactive against other CoVs than those antibodies that target S1. Additionally, a helical peptide EK1 derived from the HR2 domain of human CoV-OC43 (a strain responsible for the common cold) broadly binds to the stem region of CoVs and inhibits membrane fusion. Our goal with this supplement is to define the neutralizing epitopes on the S protein of SARS-CoV-2, such as those targeted by antibodies, the EK1 peptide and other peptides reported to bind to the RBD, to aid in both structure-based vaccine and small molecule antiviral design. Specifically, we will leverage our combined expertise in x-ray crystallography (Wilson), electron microscopy (Ward), and small-molecule discovery and medicinal chemistry (Wolan) to functionally characterize neutralization epitopes and antibody binding motifs and apply this information into high-throughput assays to aid in vaccine design, applications and use of therapeutic antibodies, and for discovery of specific high affinity small molecules to the S protein of SARS-CoV-2, as well as other coronaviruses, including SARS and MERS. We anticipate that our structural characterization of novel Abs isolated from B cells of convalescent patients will provide critical information on surface hot spots, which can be targeted by vaccines and small molecules. As such, common features employed by antibodies for epitope recognition will inform on the tailored design of compounds as lead candidates for COVID-19 antivirals. Importantly, we will subject our small molecules to biologically relevant pseudovirus plaque assays and cell-based infection models, as well as human microsomal stability assays to generate a compendium of small molecules to move forward into translational studies.

冠状病毒（Coronaviruses，CoV）是有包膜的、正义的、单链RNA病毒，并分为α和β-冠状病毒。CoV感染哺乳动物和鸟类，通常导致下呼吸道和/或上呼吸道疾病。由冠状病毒引起的人类疾病范围从普通感冒到全球流行病/大流行病，包括2003年的严重急性呼吸综合征（SARS-CoV），2004年的人类CoV-NL 63，2005年的人类CoV-HKU 1，2012年的中东呼吸综合征冠状病毒（MERS-CoV）和2019年的SARS-CoV-2。没有批准的疫苗或抗病毒药物来对抗CoV感染， 经过测试和验证的治疗方法代表了当前SARSCoV-2爆发的巨大全球关注。 2019年12月底，世界卫生组织发现中国武汉市出现异常大规模的肺炎病例聚集。在短短3个月内，全球已确诊超过100万例COVID-19确诊病例，其中近50，000例死亡。美国的数字正在以惊人的速度增长（目前约占全球总数的25%）， 死亡人数（目前约占总数的10%）。2002年，随着中国广东省首次发生SARS冠状病毒大流行以及2012年MERS冠状病毒爆发，人类冠状病毒感染的严重性和高死亡率非常明显。与SARS-CoV一样，目前的SARS-CoV-2（79%相同）也采用血管紧张素转换酶II（ACE 2）作为宿主受体进入细胞。冠状病毒表面暴露的刺突（S）蛋白负责识别和结合ACE 2，并代表了疫苗和抗病毒治疗药物开发的潜在靶点。重要的是，迄今为止从COVID-19患者中分离出的抗体（Abs）似乎结合了刺突蛋白上的几个区域，这些区域代表了小分子发现的理想靶点。 冠状病毒表面的刺突蛋白是糖基化的三聚体，由两个胞外结构域S1和S2组成。迄今为止，大多数针对CoV的nAb（中和Ab）靶向S1结构域，该结构域含有负责ACE 2结合的受体结合结构域（RBD）。尽管如此，在由茎融合机制组成的S2结构域上具有表位的Ab在基于细胞的感染模型和动物感染模型中也具有中和潜力，并且通常比靶向CoV的那些抗体对其他CoV具有更广泛的反应性。 S1.此外，衍生自人CoV-0 C43（一种引起普通感冒的毒株）的HR 2结构域的螺旋肽EK 1广泛地结合到CoV的茎区并抑制膜融合。我们的目标是确定SARS-CoV-2的S蛋白上的中和表位，例如抗体靶向的表位，EK 1肽和其他报道与RBD结合的肽，以帮助基于结构的疫苗和小分子抗病毒设计。 具体来说，我们将利用我们在X射线晶体学（威尔逊），电子显微镜（沃德）和小分子发现和药物化学（沃兰）的综合专业知识，功能性地表征中和表位和抗体结合基序，并将这些信息应用于高通量测定，以帮助疫苗设计，应用和使用治疗性抗体，以及发现对SARS-CoV-2的S蛋白以及其他冠状病毒（包括SARS和MERS）的特异性高亲和力小分子。我们预计 我们对从恢复期患者的B细胞中分离的新型Ab的结构表征将提供关于表面热点的关键信息，所述表面热点可被疫苗和小分子靶向。因此，抗体用于表位识别的共同特征将为化合物的定制设计提供信息，作为COVID-19抗病毒药物的主要候选物。重要的是，我们将对我们的小分子进行生物学相关的假病毒空斑试验和基于细胞的感染模型，以及人微粒体稳定性试验，以生成小分子的纲要，从而进入转化研究。