新型冠状病毒S蛋白RBD的糖基化及其长度对蛋白疫苗免疫原性的影响

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (12): 1645-1650.

新型冠状病毒S蛋白RBD的糖基化及其长度对蛋白疫苗免疫原性的影响

张婷, 王志荣, 许雪梅^*

中国医学科学院基础医学研究所北京协和医学院基础学院生物物理及结构生物学系, 北京 100005

收稿日期:2020-09-18 修回日期:2020-10-23 出版日期:2020-12-05 发布日期:2020-11-30
通讯作者: ^* xuemeixu@vip.sina.com; xuemeixu@ibms.pumc.edu.cn
基金资助:
中国医学科学院医学与健康科技创新工程(2020-I2M-2-014;2016-I2M-3-026)

Impact of glycosylation and length of RBD of SARS-CoV-2 S protein on the immunogenicity of RBD protein vaccines

ZHANG Ting, WANG Zhi-rong, XU Xue-mei^*

Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China

Received:2020-09-18 Revised:2020-10-23 Online:2020-12-05 Published:2020-11-30
Contact: ^* xuemeixu@vip.sina.com; xuemeixu@ibms.pumc.edu.cn

摘要/Abstract

摘要： 目的分析去糖基化及片段长度对新型冠状病毒(SARS-CoV-2)受体结合结构域(RBD)三聚体蛋白免疫原性的影响。方法以SARS-CoV-2 S蛋白的RBD为基础,分别突变敲除长度为220个氨基酸的RBD(aa.331-550)中的3个N-糖基化残基(N331、N343、N360),并分别在C端融合三聚化结构域,利用杆状病毒-昆虫细胞表达3个去糖基化的三聚体蛋白(RBDT-N1、RBDT-N2、RBDT-N3);同时构建表达2种糖基化位点不变的三聚体蛋白,即去糖基化蛋白的野生型对照RBDT和长度为273个氨基酸的RBDST(aa.319-591)。Western blot比较蛋白表达水平,亲和层析纯化后免疫小鼠,分析血清中RBD特异性IgG及SARS-CoV-2假病毒中和抗体水平。结果 RBDT-N1、RBDT-N2及RBDT-N3的表达水平均较RBDT显著提高,3种去糖基化蛋白诱发的特异性IgG及假病毒中和抗体水平均与RBDT的相当;RBDST诱发的特异性IgG抗体及假病毒中和抗体滴度较RBDT的显著提高(P＜0.05,P＜0.01)。结论去除RBD的N-糖基化残基及适当延长RBD旁侧序列有利于提高RBD蛋白疫苗的表达水平及免疫原性,研究结果为以RBD为基础的SARS-CoV-2疫苗的研发策略提供了参考。

关键词: 新型冠状病毒, 受体结合结构域, 糖基化

Abstract: Objective To analyze the impact of deglycosylation and length of RBD of SARS-CoV-2 S protein on the immunogenicity. Methods Based on the RBD of the SARS-CoV-2 S protein, three N-glycosylated residues (N331, N343, N360) in the 220 amino acid RBD (aa.331-550) were respectively mutated, and trimerization domains were fused at the C-terminus of resulted peptides. The resulted deglycosylated trimer proteins (RBDT-N1, RBDT-N2, RBDT-N3) were expressed in baculovirus-insect cell expression system. The wild-type control of deglycosylated proteins (RBDT) and RBDST (aa.319-591) with a length of 273 amino acids were also constructed. The expression levels were analyzed by Western blot. Proteins were purified by affinity chromatography. Mice were immunized, and the sera were subjected to analysis of RBD-specific IgG and neutralizing antibodies against SARS-CoV-2 pseudovirus. Results Deglycosylation increased the expression level of RBD trimers in insect cells. RBDT and three deglycosylated proteins (RBDT-N1, RBDT-N2, RBDT-N3) induced similar level of RBD-specific IgG and SARS-CoV-2 pseudovirus neutralization titers. RBDST induced high titers of RBD-specific IgG and neutralizing antibodies, which were both significantly higher than that induced by RBDT (P＜0.05,P＜0.01). Conclusions The results suggest that removing the N-glycosylation residues of RBD and extending the RBD flanking sequence are beneficial to increase the expression and immunogenicity of the RBD protein vaccine. The research provides a useful reference for the development strategy of RBD-based SARS-CoV-2 vaccines.

Key words: SARS-CoV-2, receptor binding domain, glycosylation

中图分类号:

R392

张婷, 王志荣, 许雪梅. 新型冠状病毒S蛋白RBD的糖基化及其长度对蛋白疫苗免疫原性的影响[J]. 基础医学与临床, 2020, 40(12): 1645-1650.

ZHANG Ting, WANG Zhi-rong, XU Xue-mei. Impact of glycosylation and length of RBD of SARS-CoV-2 S protein on the immunogenicity of RBD protein vaccines[J]. Basic & Clinical Medicine, 2020, 40(12): 1645-1650.

参考文献

[1] Kaur SP, Gupta V. COVID-19 vaccine: a comprehensive status report[J]. Virus Res, 2020, 288:198114. doi: 10.1016/j.virusres.2020.198114.
[2] Keech C, Albert G, Cho I, et al. Phase 1-2 trial of a SARS-CoV-2 recombinant spike protein nanoparticle vaccine[J]. N Engl J Med, 2020. doi: 10.1056/NEJMoa2026920.
[3] Xu J, Zhao S, Teng T, et al. Systematic comparison of two animal-to-human transmitted human coronaviruses: SARS-CoV-2 and SARS-CoV[J]. Viruses, 2020, 12:244-260.
[4] Walls AC, Park YJ, Tortorici MA, et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein[J]. Cell, 2020, 181:281-292.
[5] Yuan M, Wu NC, Zhu X, et al. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV[J]. Science, 2020, 368:630-633.
[6] Wang Q, Zhang Y, Wu L, et al. Structural and functional basis of SARS-CoV-2 entry by using human ACE2[J]. Cell, 2020, 181:894-904.
[7] Shajahan A, Supekar NT, Gleinich AS, et al. Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2[J]. Glycobiology, 2020. doi: 10.1093/glycob/cwaa042.
[8] Jackson LA, Anderson EJ, Rouphael NG, et al. An mRNA vaccine against SARS-CoV-2 - preliminary report[J]. N Engl J Med, 2020. doi: 10.1056/NEJMoa2022483.
[9] Wong SK, Li W, Moore MJ, et al. A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2[J]. J Biol Chem, 2004, 279:3197-3201.
[10] Du L, Zhao G, Chan CC, et al. A 219-mer CHO-expressing receptor-binding domain of SARS-CoV S protein induces potent immune responses and protective immunity[J]. Viral Immunol, 2010, 23:211-219.
[11] Wang C, Li W, Drabek D, et al. A human monoclonal antibody blocking SARS-CoV-2 infection[J]. Nat Commun, 2020, 11:1-6.
[12] Jiang S, Hillyer C, Du L. Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses[J]. Trends Immunol, 2020, 41:355-359.
[13] Chen WH, Du L, Chag SM, et al. Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate[J]. Hum Vaccines Immunother, 2014, 10:648-658.
[14] Nie J, Li Q, Wu J, et al. Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2[J]. Emerg Microbes Infect, 2020, 9:680-686.
[15] Du L, Zhao G, Chan CCS, et al. Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity[J]. Virology, 2009, 393:144-150.
[16] Yang J, Wang W, Chen Z, et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity[J]. Nature, 2020. doi: 10.1038/s41586-020-2599-8.
[17] Watanabe Y, Allen JD, Wrapp D, et al. Site-specific glycan analysis of the SARS-CoV-2 spike[J]. Science, 2020, 369:330-333.
[18] Li Q, Wu J, Nie J, et al. The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity[J]. Cell, 2020, 182:1284-1294.
[19] Dai L, Zheng T, Xu K, et al. A universal design of betacoronavirus vaccines against COVID-19, MERS, and SARS[J]. Cell, 2020, 182:722-733.
[20] Shen C, Wang Z, Zhao F, et al. Treatment of 5 critically Ⅲ patients with COVID-19 with convalescent plasma[J]. JAMA, 2020, 323:1582-1589.
[21] Pooladanda V, Thatikonda S, Godugu C. The current understanding and potential therapeutic options to combat COVID-19[J]. Life Sci, 2020, 254:117765. doi: 10.1016/j.lfs.2020.117765.
[22] Zost SJ, Gilchuk P, Case JB, et al. Potently neutralizing and protective human antibodies against SARS-CoV-2[J]. Nature, 2020, 584:443-449.