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| 严重急性呼吸综合征冠状病毒2 DNA疫苗与重组亚单位疫苗在小鼠中诱导中和抗体的效力分析 |
| 徐铮昊1△,王诚2△,余润芷2,丁翠玲1,何燕华1,江亮亮1,彭浩然1,吴俊杰3,赵平1,戚中田1* |
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(1. 海军军医大学(第二军医大学)海军医学系生物医学防护教研室, 上海 200433;
2. 海军军医大学(第二军医大学)基础医学院学员八队, 上海 200433;
3. 海军军医大学(第二军医大学)长海医院呼吸与危重症医学科, 上海 200433
△共同第一作者
*通信作者) |
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| 摘要: |
| 目的 探讨以严重急性呼吸综合征冠状病毒2（SARS-CoV-2）受体结合区（RBD）和表面刺突蛋白（S蛋白）S1亚基为疫苗靶抗原诱导中和抗体的效果。方法 构建SARS-CoV-2 RBD与小鼠IgG1 Fc段（mFc）融合蛋白表达质粒pVRC-RBD-mFc，转染人胚肾细胞293T并进行培养。用蛋白质印迹法检测细胞培养上清中的RBD-mFc融合蛋白，用微量中和实验检测细胞培养上清中的RBD-mFc及CHO细胞重组表达的SARS-CoV-2 S1与人IgG1 Fc段（S1-hFc）融合蛋白对SARS-CoV-2感染的抑制作用。分别用质粒pVRC-RBD-mFc及S1-hFc融合蛋白通过肌内注射接种BALB/c小鼠，用ELISA检测小鼠血清中的抗-S1 IgG，用微量中和实验检测小鼠血清的病毒中和活性。结果 pVRC-RBD-mFc质粒转染293T细胞的培养上清中可检测到RBD-mFc融合蛋白，超滤浓缩的细胞培养上清及S1-hFc融合蛋白均呈浓度依赖性抑制SARS-CoV-2对Vero E6细胞的感染；经pVRC-RBD-mFc质粒及S1-hFc融合蛋白免疫的小鼠血清中均可检测出抗-S1 IgG，且能中和SARS-CoV-2的感染；S1-hFc融合蛋白免疫小鼠血清的抗体滴度及病毒中和活性均高于质粒pVRC-RBD-mFc免疫小鼠血清（P均<0.01）。结论 SARS-CoV-2 RBD和S1蛋白均可能作为有效的疫苗抗原，重组亚单位疫苗较DNA疫苗能更有效地诱导中和抗体。 |
| 关键词: 严重急性呼吸综合征冠状病毒2 DNA疫苗 亚单位疫苗 中和抗体 |
| DOI：10.16781/j.0258-879x.2020.05.0474 |
| 投稿时间：2020-04-30修订日期：2020-05-19 |
| 基金项目:国家重点研发计划（2016YFC1200401），国家重大科技专项（2017ZX10304403-003）. |
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| Efficacy analysis of severe acute respiratory syndrome coronavirus 2 DNA vaccine and recombinant subunit vaccine inducing neutralizing antibodies in mice |
| XU Zheng-hao1△,WANG Cheng2△,YU Run-zhi2,DING Cui-ling1,HE Yan-hua1,JIANG Liang-liang1,PENG Hao-ran1,WU Jun-jie3,ZHAO Ping1,QI Zhong-tian1* |
(1. Department of Biomedical Defense, Faculty of Naval Medicine, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. The Eighth Student Team, College of Basic Medical Sciences, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
3. Department of Respiratory and Critical Care Medicine, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China
△Co-first authors.
* Corresponding author) |
| Abstract: |
| Objective To investigate the efficacy of neutralizing antibodies induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) and spike (S) protein S1 subunit. Methods The SARS-CoV-2 RBD and mouse immunoglobulin G1 (IgG1) Fc fragment (mFc) fusion protein expression plasmid pVRC-RBD-mFc was constructed and transfected into human embryonic kidney 293T cells. The RBD-mFc fusion protein in the cell supernatants was detected by Western blotting. The effect of RBD-mFc in cell supernatants and CHO recombinant S1-human IgG1 Fc (S1-hFc) fusion protein on SARS-CoV-2 infection was detected by microneutralization test. BALB/c mice were immunized with plasmid pVRC-RBD-mFc and S1-hFc fusion protein via intramuscular injection. Anti-S1 IgG antibodies in mouse sera were detected by enzyme-linked immunosorbent assay (ELISA), and the virus neutralization activity of mouse sera was detected by microneutralization test. Results The RBD-mFc fusion protein could be detected in the culture supernatants of 293T cells transfected with the plasmid pVRC-RBD-mFc, the concentrated supernatants and the S1-hFc fusion protein could inhibit SARS-CoV-2 infection on Vero E6 cells in a concentration-dependent manner. Anti-S1 IgG antibodies could be detected in the sera of mice immunized with plasmid pVRC-RBD-mFc and S1-hFc fusion protein, and the sera of both groups could neutralize SARS-CoV-2 infection. The serum antibody titers and virus neutralization activity of S1-hFc fusion protein immunized mice were significantly higher than those of plasmid pVRC-RBD-mFc immunized mice (both P<0.01). Conclusion Both SARS-CoV-2 RBD and S1 subunit may be used as effective vaccine antigens. Compared with DNA vaccine, recombinant subunit vaccine can induce neutralizing antibody more effectively. |
| Key words: severe acute respiratory syndrome coronavirus 2 DNA vaccines subunit vaccines neutralizing antibodies |
