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

Efficacy analysis of severe acute respiratory syndrome coronavirus 2 DNA vaccine and recombinant subunit vaccine inducing neutralizing antibodies in mice

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。

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 pVRCRBD-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.