Table_1_Monoclonal antibodies constructed from COVID-19 convalescent memory B cells exhibit potent binding activity to MERS-CoV spike S2 subunit and other human coronaviruses.doc

IntroductionThe Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are two highly contagious coronaviruses causing MERS and COVID-19, respectively, without an effective antiviral drug and a long-lasting vaccine. Approaches for diagnosis, therapeutics, prevention, etc., particularly for SARS-CoV-2 that is continually spreading and evolving, are urgently needed. Our previous study discovered that >60% of sera from convalescent COVID-19 individuals, but <8% from general population, showed binding activity against the MERS-CoV spike protein, indicating that SARS-CoV-2 infection boosted antibodies cross-reactive with MERS-CoV.
MethodsTo generate antibodies specific to both SARS-CoV-2 and MERS-CoV, here we screened 60 COVID-19 convalescent sera against MERS-CoV spike extracellular domain and S1 and S2 subunits. We constructed and characterized monoclonal antibodies (mAbs) from COVID-19 convalescent memory B cells and examined their binding and neutralizing activities against human coronaviruses.
Results and DiscussionOf 60 convalescent serum samples, 34 showed binding activity against MERS-CoV S2, with endpoint titers positively correlated with the titers to SARS-CoV-2 S2. By sorting single memory B cells from COVID-19 convalescents, we constructed 38 mAbs and found that 11 mAbs showed binding activity with MERS-CoV S2, of which 9 mAbs showed potent cross-reactivity with all or a proportion of spike proteins of alphacoronaviruses (229E and NL63) and betacoronaviruses (SARS-CoV-1, SARS-CoV-2, OC43, and HKU1). Moreover, 5 mAbs also showed weak neutralization efficiency against MERS-CoV spike pseudovirus. Epitope analysis revealed that 3 and 8 mAbs bound to linear and conformational epitopes in MERS-CoV S2, respectively. In summary, we have constructed a panel of antibodies with broad-spectrum reactivity against all seven human coronaviruses, thus facilitating the development of diagnosis methods and vaccine design for multiple coronaviruses.

**引言**
中东呼吸综合征冠状病毒（Middle East respiratory syndrome coronavirus, MERS-CoV）与新型冠状病毒（severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）均为高传染性冠状病毒，分别引发中东呼吸综合征与新型冠状病毒肺炎（COVID-19），目前尚无特效抗病毒药物与长效疫苗。针对此类冠状病毒的诊断、治疗与防控等手段亟待开发，其中对于持续传播并不断变异的SARS-CoV-2而言，相关研究需求尤为迫切。本团队前期研究发现，超过60%的新冠康复者血清可与MERS-CoV刺突蛋白结合，而普通人群中该阳性率不足8%，提示SARS-CoV-2感染可诱导产生与MERS-CoV具有交叉反应性的抗体。

**方法**
为获得同时针对SARS-CoV-2与MERS-CoV的特异性抗体，本研究以MERS-CoV刺突蛋白胞外结构域及其S1、S2亚基为靶标，对60份新冠康复者血清进行筛选。我们从新冠康复者的记忆B细胞中制备并鉴定单克隆抗体（monoclonal antibodies, mAbs），并检测这些抗体针对人类冠状病毒的结合活性与中和活性。

**结果与讨论**
在60份康复者血清样本中，共有34份可与MERS-CoV S2蛋白结合，其终点滴度与针对SARS-CoV-2 S2蛋白的滴度呈正相关。通过分选新冠康复者的单个记忆B细胞，我们成功制备了38株单克隆抗体，其中11株可与MERS-CoV S2蛋白结合；在这些抗体中，9株对α冠状病毒（229E与NL63）及β冠状病毒（SARS-CoV-1、SARS-CoV-2、OC43与HKU1）的全部或部分刺突蛋白表现出强效交叉反应性。此外，另有5株抗体对MERS-CoV刺突蛋白假病毒展现出微弱的中和活性。表位分析结果显示，分别有3株与8株抗体靶向结合MERS-CoV S2蛋白的线性表位与构象表位。综上，本研究构建了一组可针对全部7种人类冠状病毒的广谱反应性抗体，可为多种冠状病毒的诊断方法开发与疫苗设计提供有力支持。