Guo Yan, He Wenhui, Mou Huihui, Zhang Lizhou, Chang Jing, Peng Shoujiao, Ojha Amrita, Tavora Rubens, Parcells Mark S, Luo Guangxiang, Li Wenhui, Zhong Guocai, Choe Hyeryun, Farzan Michael, Quinlan Brian D
Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida, USA.
Department of Animal and Food Sciences, University of Delaware, Newark, Delaware, USA.
mBio. 2021 May 11;12(3):e00930-21. doi: 10.1128/mBio.00930-21.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines. All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突（S）蛋白介导病毒进入表达血管紧张素转换酶2（ACE2）的细胞。S蛋白通过其受体结合域（RBD）与ACE2结合，RBD是1273个氨基酸的S蛋白原体中一个独立折叠的197个氨基酸片段。RBD是SARS-CoV-2主要的中和表位，也是任何SARS-CoV-2疫苗的关键靶点。在此，我们表明，与两种载体蛋白之一偶联的这种RBD在免疫的啮齿动物中引发的中和反应比类似偶联的脯氨酸稳定化S蛋白胞外域更有效。然而，天然RBD表达效率低下，限制了其作为疫苗抗原的用途。不过，我们表明，工程化的带有四个新糖基化位点的RBD（gRBD）表达效率显著更高，并且作为DNA疫苗抗原比野生型RBD或全长S蛋白产生更有效的中和反应，尤其是当与多价载体（如铁蛋白24聚体）融合时。此外，当作为亚单位蛋白疫苗给药时，gRBD比野生型RBD更具免疫原性。我们的数据表明，多价gRBD抗原可以降低所有主要类型SARS-CoV-2疫苗的成本和剂量，并提高其免疫原性。所有现有的2019冠状病毒病（COVID-19）疫苗都表达或递送全长SARS-CoV-2刺突（S）蛋白。我们表明，这种抗原并非最佳选择，这与绝大多数针对该病毒的中和反应集中在S蛋白受体结合域（RBD）的观察结果一致。然而，这种RBD作为一个独立结构域表达不佳，尤其是当与多价支架融合表达时。因此，我们通过在RBD通常被全长S蛋白遮挡的一面引入四个糖基化位点，构建了一种表达水平更高的SARS-CoV-2 RBD形式。我们表明，这种工程化蛋白gRBD在基因疫苗和蛋白递送疫苗中都比野生型RBD或全长S蛋白更具免疫原性。
