DataSheet_1_Antigenic Cross-Reactivity Between SARS-CoV-2 S1-RBD and Its Receptor ACE2.docx

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus responsible for the ongoing COVID-19 pandemic. SARS-CoV-2 binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain in the S1 subunit of the spike protein (S1-RBD). The serum levels of autoantibodies against ACE2 are significantly higher in patients with COVID-19 than in controls and are associated with disease severity. However, the mechanisms through which these anti-ACE2 antibodies are induced during SARS-CoV-2 infection are unclear. In this study, we confirmed the increase in antibodies against ACE2 in patients with COVID-19 and found a positive correlation between the amounts of antibodies against ACE2 and S1-RBD. Moreover, antibody binding to ACE2 was significantly decreased in the sera of some COVID-19 patients after preadsorption of the sera with S1-RBD, which indicated that antibodies against S1-RBD can cross-react with ACE2. To confirm this possibility, two monoclonal antibodies (mAbs 127 and 150) which could bind to both S1-RBD and ACE2 were isolated from S1-RBD-immunized mice. Measurement of the binding affinities by Biacore showed these two mAbs bind to ACE2 much weaker than binding to S1-RBD. Epitope mapping using synthetic overlapping peptides and hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that the amino acid residues P463, F464, E465, R466, D467 and E471 of S1-RBD are critical for the recognition by mAbs 127 and 150. In addition, Western blotting analysis showed that these mAbs could recognize ACE2 only in native but not denatured form, indicating the ACE2 epitopes recognized by these mAbs were conformation-dependent. The protein–protein interaction between ACE2 and the higher affinity mAb 127 was analyzed by HDX-MS and visualized by negative-stain transmission electron microscopy imaging combined with antigen-antibody docking. Together, our results suggest that ACE2-cross-reactive anti-S1-RBD antibodies can be induced during SARS-CoV-2 infection due to potential antigenic cross-reactivity between S1-RBD and its receptor ACE2.

严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）是一种新兴病毒，引发了持续的COVID-19大流行。SARS-CoV-2通过其刺突蛋白S1亚基的受体结合域（S1-RBD）与人类细胞受体血管紧张素转换酶2（ACE2）结合。与对照者相比，COVID-19患者的血清中针对ACE2的自身抗体水平显著升高，并且与疾病严重程度相关。然而，这些抗ACE2抗体在SARS-CoV-2感染期间诱导的机制尚不明确。在本研究中，我们证实了COVID-19患者中ACE2抗体的增加，并发现ACE2抗体与S1-RBD的抗体量之间存在正相关。此外，在用S1-RBD预先吸附血清后，一些COVID-19患者的血清中ACE2的结合显著降低，这表明针对S1-RBD的抗体可以与ACE2发生交叉反应。为了证实这一可能性，我们从S1-RBD免疫小鼠中分离出两种可以结合S1-RBD和ACE2的单克隆抗体（mAbs 127和150）。通过Biacore测定的结合亲和力显示，这两种mAbs与ACE2的结合亲和力远低于与S1-RBD的结合亲和力。使用合成重叠肽和氢氘交换质谱分析（HDX-MS）进行表位映射揭示了S1-RBD中氨基酸残基P463、F464、E465、R466、D467和E471对于mAbs 127和150的识别至关重要。此外，通过Western blotting分析显示，这些mAbs只能在天然形式下识别ACE2，而不能在变性形式下识别，这表明这些mAbs识别的ACE2表位是构象依赖性的。通过HDX-MS分析了ACE2与更高亲和力mAb 127之间的蛋白质-蛋白质相互作用，并使用负染透射电子显微镜成像结合抗原-抗体对接进行可视化。总之，我们的研究结果提示，在SARS-CoV-2感染期间，由于S1-RBD与其受体ACE2之间潜在的抗原交叉反应，可能会诱导ACE2交叉反应性抗S1-RBD抗体。