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年在中国武汉出现的新型人β冠状病毒SARS-CoV 2迅速演变 变成了全球性的流行病超过1亿人被感染， 死亡COVID-19疾病的表现方式也存在很大差异，从无症状到无症状， 感染致死。与成人相比，儿童COVID-19疾病总体上更为轻微或无症状。一 可以解释这种差异的假设是，儿童对SARS-CoV 2具有交叉免疫，这是由于儿童经常 早期暴露于全球传播的人类冠状病毒（HCoV），导致轻度呼吸道疾病。 地方性HCoV和SARS-CoV 2之间是否存在某种程度的交叉免疫， 成年期，并能提供一定程度的COVID-19疾病的保护是本R 01申请的主题。 我们的主要目标是提供抗HCoV/SARS-CoV 2刺突交叉的血清学和分子证据， 反应性和中和性抗体，可以在体内提供针对SARS-CoV 2的保护。我们有内部调查委员会- 批准了对250名COVID-19患者采集血液样本的方案。我们的COVID队列由5个 包括成人和儿童癌症患者、成人和儿童医疗保健提供者以及成人 不接触新冠肺炎患者。此外，我们将研究大流行前的季节性流感队列， 已有抗SARS-CoV 2 S抗体的证据。在目标1中，我们将定量抗S HCoV抗体的存在 并定量其与SARS-CoV 2 S的交叉反应性。子目标1A中的研究将包括FACS染色， S表达细胞和用于表位作图的S亚结构域的ELISA。在子目标1B中，选择的血浆样本 将用于血浆IgG的亲和柱纯化， 并从4个HCoV或SARS-CoV 2加标柱之一洗脱，并测试交叉结合、交叉Fc效应子 活性和交叉中和活性。在子目标1C中，将在hACE 2小鼠中体内测试这些纯化的IgG 对SARS-CoV 2攻击的交叉保护。在目标2中，我们将建立双- CoV之间的定向免疫可以通过共同的免疫途径提供对HCoV和SARS-CoV 2的交叉免疫。 刺突表位识别。在子目标2A中，我们将进行记忆B（mB）细胞筛选，以确定S交叉的存在。 约束力结合至少一种hCoV S蛋白和SARS-CoV 2 S的单个mB细胞将通过FACS分离，其 克隆同源VH/VL基因，表达为完整IgG 1 mAb，并检测交叉结合、病毒中和 和针对不同HoCoV、SARS和SARS-CoV 2的Fc效应子活性。在子目标2B中，具有交叉- 将在小鼠和仓鼠模型中测试CoV活性，以保护免受SARS-CoV 2攻击。在 在子目标2C中，我们将采用新的LibraSeq技术来捕获单个或多个刺突结合特异性， 选择Bm细胞的BCR库和转录组，以研究潜在的不同进化起源， 可能存在于单刺突和多刺突结合细胞之间。此R 01赠款将提供原则证明 HCoV/SARS-CoV 2 Ab交叉免疫的分子研究，可能有助于COVID-19疫苗设计。

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.