The ongoing COVID-19 pandemic caused by SARS-CoV-2 is a huge public health crisis for the globe. The receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein plays a vital role in viral infection and serves as a major target for developing neutralizing antibodies. In this study, the antibody response to the RBD of SARS-CoV-2 S protein was analyzed by a panel of sera from animals immunized with RBD-based antigens and four linear B-cell epitope peptides (R345, R405, R450 and R465) were revealed. The immunogenicity of three immunodominant peptides (R345, R405, R465) was further accessed by peptide immunization in mice, and all of them could induced potent antibody response to SARS-CoV-2 S protein, indicating that the three determinants in the RBD were immunogenic. We further generated and characterized monoclonal antibodies (15G9, 12C10 and 10D2) binding to these epitope peptides, and finely mapped the three immunodominant epitopes using the corresponding antibodies. Neutralization assays showed that all three monoclonal antibodies had neutralization activity. Results from IFA and western blotting showed that 12C10 was a cross-reactive antibody against both of SARS-CoV-2 and SARS-CoV. Results from conservative and structural analysis showed that ³⁵⁰VYAWN³⁵⁴ was a highly conserved epitope and exposed on the surface of SARS-CoV-2 S trimer, whereas ⁴⁷³YQAGSTP⁴⁷⁹ located in the receptor binding motif (RBM) was variable among different SARS-CoV-2 strains. ⁴⁰⁷VRQIAP⁴¹² was a highly conserved, but cryptic epitope shared between SARS-CoV-2 and SARS-CoV. These findings provide important information for understanding the humoral antibody response to the RBD of SARS-CoV-2 S protein and may facilitate further efforts to design SARS-CoV-2 vaccines and the target of COVID-19 diagnostic.

由 SARS-CoV-2 引起的持续的 COVID-19 大流行对全球来说是一场巨大的公共卫生危机。SARS-CoV-2 刺突 (S) 蛋白的受体结合域 (RBD) 在病毒感染中起着至关重要的作用，并且是开发中和抗体的主要目标。在这项研究中，通过一组用基于 RBD 的抗原和四种线性 B 细胞表位肽（R345、R405、R450 和 R465）免疫的动物的血清分析了 SARS-CoV-2 S 蛋白对 RBD 的抗体反应被揭露。三种免疫显性肽（R345、R405、R465）的免疫原性通过小鼠肽免疫进一步获得，它们都可以诱导针对SARS-CoV-2 S蛋白的强效抗体反应，表明RBD中的三个决定簇具有免疫原性. 我们进一步生成并表征了单克隆抗体（15G9、12C10 和 10D2) 结合这些表位肽，并使用相应的抗体精细定位三个免疫优势表位。中和试验表明所有三种单克隆抗体都具有中和活性。IFA 和蛋白质印迹的结果表明，12C10 是一种针对 SARS-CoV-2 和 SARS-CoV 的交叉反应抗体。保守和结构分析的结果表明，³⁵⁰ VYAWN ³⁵⁴是高度保守的表位，暴露在 SARS-CoV-2 S 三聚体的表面，而位于受体结合基序 (RBM) 中的⁴⁷³ YQAGSTP ⁴⁷⁹在不同 SARS-CoV-2 菌株之间是可变的。⁴⁰⁷ VRQIAP ⁴¹²是 SARS-CoV-2 和 SARS-CoV 之间共享的高度保守但隐蔽的表位。这些发现为理解 SARS-CoV-2 S 蛋白对 RBD 的体液抗体反应提供了重要信息，并可能有助于进一步努力设计 SARS-CoV-2 疫苗和 COVID-19 诊断目标。