DataSheet1_In Planta Production of the Receptor-Binding Domain From SARS-CoV-2 With Human Blood Group A Glycan Structures.pdf

Glycosylation of viral envelope proteins is important for infectivity and immune evasion. The SARS-CoV-2 spike protein is heavily glycosylated and host-derived glycan modifications contribute to the formation of specific immunogenic epitopes, enhance the virus-cell interaction or affect virus transmission. On recombinant viral antigens used as subunit vaccines or for serological assays, distinct glycan structures may enhance the immunogenicity and are recognized by naturally occurring antibodies in human sera. Here, we performed an in vivo glycoengineering approach to produce recombinant variants of the SARS-CoV-2 receptor-binding domain (RBD) with blood group antigens in Nicotiana benthamiana plants. SARS-CoV-2 RBD and human glycosyltransferases for the blood group ABH antigen formation were transiently co-expressed in N. benthamiana leaves. Recombinant RBD was purified and the formation of complex N-glycans carrying blood group A antigens was shown by immunoblotting and MS analysis. Binding to the cellular ACE2 receptor and the conformation-dependent CR3022 antibody showed that the RBD glycosylation variants carrying blood group antigens were functional. Analysis of sera from RBD-positive and RBD-negative individuals revealed further that non-infected RBD-negative blood group O individuals have antibodies that strongly bind to RBD modified with blood group A antigen structures. The binding of IgGs derived from sera of non-infected RBD-negative blood group O individuals to blood group A antigens on SARS-CoV-2 RBD suggests that these antibodies could provide some degree of protection from virus infection.

病毒包膜蛋白的糖基化对于感染性和免疫逃逸至关重要。SARS-CoV-2的刺突蛋白高度糖基化，宿主来源的糖基化修饰有助于形成特定的免疫原性表位，增强病毒与细胞的相互作用或影响病毒的传播。在作为亚单位疫苗或用于血清学检测的重组病毒抗原中，独特的糖基结构可能增强免疫原性，并被人血清中自然存在的抗体所识别。本研究中，我们采用体内糖基工程方法，在 Nicotiana benthamiana 植物中生产了携带血型抗原的 SARS-CoV-2 受体结合域（RBD）的重组变异体。SARS-CoV-2 RBD 和用于血型 ABH 抗原形成的人类糖基转移酶在 N. benthamiana 叶片中瞬时共表达。通过纯化重组 RBD，并通过免疫印迹和质谱分析证实了携带血型 A 抗原的复杂 N-糖基的形成。RBD 与细胞 ACE2 受体的结合以及与构象依赖性 CR3022 抗体的结合表明，携带血型抗原的 RBD 糖基化变异体具有功能。对 RBD 阳性和 RBD 阴性个体血清的分析进一步揭示，未感染且 RBD 阴性的血型 O 个体具有与血型 A 抗原结构修饰的 RBD 强烈结合的抗体。来自未感染 RBD 阴性血型 O 个体血清的 IgGs 与 SARS-CoV-2 RBD 上的血型 A 抗原的结合表明，这些抗体可能在一定程度上提供对病毒感染的防护。