Serologic and Molecular Studies of human anti-hCoV antibody cross-immunity and protective responses among endemic HCoVs and SARS-CoV2

项目摘要 2019年武汉出现新型人β-冠状病毒SARS-CoV2中国迅速进化 变成一场世界性的大流行。已有超过1亿人被感染，有数百万人 死亡。新冠肺炎病的表现方式也与无症状的有很大差异 感染致死。与成人相比，儿童新冠肺炎疾病总体上更轻微或更无症状。一 可以解释这种差异的假设是，由于儿童频繁地感染SARS-CoV2，他们对SARS-CoV2有交叉免疫 早期接触全球流行的人类冠状病毒(HCoV)，会导致较轻微的呼吸道疾病。 地方性HCoV和SARS-CoV2之间是否存在某种程度的交叉免疫 成年并能对新冠肺炎病提供一定程度的保护是这款R01应用程序的主题。 我们的主要目标是提供抗HCoV/SARS-CoV2刺突(S)交叉的血清学和分子证据。 反应性和中和性抗体，可在体内提供对SARS-CoV2的保护。我们有一个内部审查委员会- 批准了对250名新冠肺炎患者采集血液样本的协议。我们的COVID队列由5人组成 包括成人和儿童癌症患者、成人和儿童保健提供者以及成年人的群体 不接触新冠状病毒患者。此外，我们将研究大流行前的季节性流感队列，以 先前存在的抗SARS-CoV2 S抗体的证据。在目标1中，我们将对抗S HCoV抗体的存在进行量化 并对它们与SARS-CoV2 S的交叉反应性进行定量。 用于表位定位的S表达细胞和S亚区的ELISA。在SubAim 1B中，选定的血浆样本 在每个研究组内，将用于亲和层析纯化将通过的血浆IgG 从4个HCoV或SARS-CoV2尖状柱中的一个中洗脱并测试交叉结合、交叉Fc效应 活性和交叉中和活性。在SubAim 1C中，这些纯化的免疫球蛋白将在hACE2小鼠身上进行体内测试 针对SARS-CoV2挑战的交叉保护。在目标2中，我们将建立分子基础，通过它，双- 针对冠状病毒间的定向免疫可通过共同的 尖峰表位识别。在子目标2A中，我们将进行记忆B(MB)细胞的筛选，以确定S是否存在交叉感染。 有约束力的。与至少一个人冠状病毒S蛋白和SARS-CoV2 S结合的单个MB细胞将通过流式细胞仪分离，其 克隆同源VH/VL基因，表达完整的IgG1单抗，并进行交叉结合、病毒中和试验 对不同的HoCoV、SARS和SARS-CoV2的Fc效应活性。在亚目的2B中，有交叉的单抗- 冠状病毒活性将在小鼠和仓鼠模型中进行测试，以防止SARS-CoV2挑战。在……里面 子目标2C，我们将采用新的LibraSeq技术来捕捉单或多个尖峰结合的特异性， 选择BM细胞的BCR谱系和转录本，研究潜在的不同进化起源 可能存在于单穗和多穗结合细胞之间。这笔R01拨款将提供原则证明 HCoV/SARS-CoV2Ab交叉免疫的分子研究可能有助于新冠肺炎疫苗的设计。

Project Summary The emergence of the novel human betacoronavirus SARS-CoV2 in Wuhan, China in 2019 has rapidly evolved into a worldwide pandemic. Over a 100 million people have been infected and there have been several million deaths. There is also great disparity in the manner in which COVID-19 illness presents, from asymptomatic infection to death. COVID-19 illness in children is overall more mild or asymptomatic compared to adults. One hypothesis that may explain this disparity is that children have cross-immunity to SARS-CoV2 due to frequent early exposure to globally circulating human coronaviruses (HCoVs) that cause a milder respiratory illness. Whether there is some level of cross-immunity between the endemic HCoVs and SARS-CoV2 that carries into adulthood and can provide some level of protection from COVID-19 disease is the subject of this R01 application. Our primary goal is to provide serologic and molecular evidence of anti-HCoV/SARS-CoV2 spike (S) cross- reactive and neutralizing antibodies that can provide protection against SARS-CoV2 in vivo. We have an IRB- approved protocol to collect blood samples on 250 COVID-19 individuals. Our COVID cohort is comprised of 5 groups that includes adult and pediatric cancer patients, adult and pediatric healthcare providers and adults without COVID patient contact. In addition, we will study our pre-pandemic seasonal influenza cohort for evidence of pre-existing anti-SARS-CoV2 S Abs. In Aim 1 we will quantify the present of anti-S HCoV antibodies and quantitate their cross-reactivity to SARS-CoV2 S. The studies in subaim 1A will include FACS staining of S expressing cells and ELISAs of S subdomains for epitope mapping. In subaim 1B, selected plasma samples within each study group will be used for affinity column purification of plasma IgGs that will be passaged over and eluted from one of 4 HCoV or SARS-CoV2 spike columns and tested for cross-binding, cross-Fc effector activity and cross-neutralization activity. In subaim 1C, these purified IgGs will be tested in vivo in hACE2 mice for cross-protection against SARS-CoV2 challenge. In Aim 2 we will establish the molecular basis by which bi- directional immunity to among CoVs could provide cross immunity to HCoVs and SARS-CoV2 through common spike epitope recognition. In subaim 2A, we will perform memory B (mB) cell screening for presence of S cross- binding. Single mB cells that bind at least one hCoV S protein and SARS-CoV2 S will be isolated by FACS, their cognate VH/VL genes cloned, expressed as whole IgG1 mAbs and tested for cross-binding, virus neutralization and Fc effector activity against the different HoCoVs, SARS and SARS-CoV2. In subaim 2B, mAbs with cross- CoV activity will be tested in mouse and hamster models for protection against SARS-CoV2 challenge. In subaim 2C, we will adapt the novel LibraSeq technique to capture the single or multi-spike binding specificity, BCR repertoires and transcriptomes of selected Bm cells to study the potential different evolutionary origins that may exist between mono-spike and multi-spike binding cells. This R01 grant will provide proof-of-principle molecular studies of HCoV/SARS-CoV2 Ab cross-immunity that may aid in COVID-19 vaccine design.