T细胞表位对新型冠状病毒疫苗诱导抗体反应的影响

Effect of T cell epitopes on antibody response induced by SARS-CoV-2 vaccine

目的通过构建严重急性呼吸系统综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)S1蛋白与通用型辅助性T淋巴细胞表位(pan HLA DR-binding epitope,PADRE)融合的DNA疫苗,探讨PADRE对S1蛋白DNA疫苗诱导抗体水平的影响。方法以pJW4303质粒为载体分别构建表达S1蛋白的重组质粒(pJW4303-S1)、表达S1和PADRE融合蛋白的重组质粒(pJW4303-S1-PADRE)。以人源密码子优化的SARS-CoV-2刺突蛋白基因为模板设计引物,PCR扩增获得基因片段,并将其克隆入真核表达载体pJW4303。对pJW4303-S1、pJW4303-S1-PADRE进行PCR扩增和酶切,产物通过琼脂糖凝胶电泳验证,并将重组质粒进行测序。构建正确的质粒转染293T细胞,Western blot验证蛋白体外表达情况。以C57BL/6小鼠为实验动物并分组,采用不同的免疫接种策略进行动物免疫,每次免疫间隔2周,免疫前采血,第6次免疫2周后处死小鼠并采血。通过ELISA检测不同免疫策略诱导的针对SARS-CoV-2 S蛋白受体结合域(receptor binding domain,RBD)的特异性抗体水平及其亲和力指数。结果PCR扩增和酶切产物的琼脂糖凝胶电泳结果及质粒测序结果表明质粒构建成功;Western blot结果表明这2个质粒可以在体外稳定表达S1蛋白、S1融合PADRE蛋白;ELISA结果显示,pJW4303-S1、pJW4303-S1-PADRE单次免疫均可以诱导产生明显的针对RBD的抗体水平(P<0.0001,P=0.0034)。不同的免疫策略在进行6次免疫后,针对RBD的特异性抗体水平在各组间差异无统计学意义(P>0.05)。抗体亲和力检测结果显示,第6次免疫后不同免疫策略诱导的针对RBD的抗体亲和力指数差异也无统计学意义(P>0.05)。结论pJW4303-S1、pJW4303-S1-PADRE均具有较强的体液免疫原性,但PADRE不能进一步提高S1蛋白DNA疫苗的体液免疫原性。

Objective We constructed a DNA vaccine fused with SARS-CoV-2 S1 protein and pan HLA DR-binding epitope(PADRE)to investigate the effect of PADRE on the level of antibodies induced by the S1 protein DNA vaccine.Methods A recombinant plasmid expressing S1 protein(pJW4303-S1)and a recombinant plasmid expressing S1 and PADRE fusion protein(pJW4303-S1-PADRE)were constructed using the pJW4303 plasmid as a vector.Primers containing PADRE were designed using SARS-CoV-2 S protein gene optimized by human codon as template.The gene fragment was amplified by PCR and cloned into the eukaryotic expression vector pJW4303.PCR amplification and enzyme digestion were performed for pJW4303-S1 and pJW4303-S1-PADRE,the products were verified by agarose gel electrophoresis,and the recombinant plasmid was sequenced.The correct plasmid was constructed and transfected with 293T cells,and the protein expression was verified by western blot in vitro.C57BL/6 mice were used as experimental animals and grouped.Different immunization strategies were used to immunize animals.Blood samples were collected before immunization and two weeks after the sixth immunization.Specific antibody levels against SARS-CoV-2 S protein receptor-binding domain(RBD)induced by different immune strategies were detected by ELISA.Results The results of agarose gel electrophoresis of PCR amplification and digestion products and plasmid sequencing indicated successful plasmid construction;Western blot results showed that S1 protein and S1 fused PADRE protein could be expressed stably in vitro;ELISA results showed that single immunization with pJW4303-S1 and pJW4303-S1-PADRE could induce significant levels of antibody against RBD(P<0.0001,P=0.0034).There was no difference in RBD-specific antibody levels between groups after 6 immunizations with different immunization strategies(P>0.05).The results of antibody affinity test showed that there was no difference in the affinity index of antibodies against RBD induced by different immunization strategies after the sixth immunization(P>0.05).Conclusion pJW4303-S1 and pJW4303-S1-PADRE had strong humoral immunogenicity,but PADRE could not further improve the humoral immunogenicity of S1 protein DNA vaccine.