C3d对分泌型柯萨奇病毒B组3型VP1 DNA疫苗的免疫增强作用

C3d enhances immune response to the secreted form of Coxsackie virus B3 VP1 DNA vaccine

目的构建分泌型柯萨奇病毒 B 组3型(CVB3)衣壳蛋白1(sVP1)与三拷贝补体3片段(C3d3)融合基因疫苗,并观察其免疫效果。方法将 C3d3 cDNA 与带有白细胞介素2(IL-2)信号肽的 CVB3 VP1基因拼接,构建真核表达质粒 pcDNA3/sVP1-C3d3。BALB/c 小鼠随机分为3组,分别肌肉注射 pcDNA3/sVP1-C3d3、pcDNA3/sVP1和 pcDNA3质粒,于不同时间检测小鼠血清中和抗体滴度、特异性细胞毒性 T 淋巴细胞(CTL)杀伤活性、血中病毒滴度,并观察疫苗的免疫保护作用。结果小鼠的中和抗体滴度随免疫次数增加而提高,第3次免疫后 pcDNA3/sVP1-C3d3组中和抗体滴度(33.6±1.7)和特异性 CTL 杀伤率(66.1%±2.9%)均明显高于 pcDNA3/sVP1组(28.3±1.7,52.8%±3.3%,均 P<0.05);以致死量 CVB3感染小鼠后,经融合基因免疫的小鼠血中病毒滴度显著降低,生存率达50%,而 pcDNA3/sVP1组仅为25%,pcDNA3组无存活。结论 C3d 可以显著增强sVP1基因免疫诱导的特异性免疫应答。

Objective To explore a strategy to enhance the specific immune response induced by secreted form of Coxsackie virus B3 （CVB3） capsid protein 1 （ sVPI ） gene vaccination against Coxsackie virus infection. Methods Eukaryotic expression plasmid pcDNA3/sVPI-C3d3 was constructed by conjugating the 3 copies of C3d （C3d3） with the secreted form of sVPI of CVB3. 42 BALB/c mice were randomly divided into 3 equal groups to be inoculated with pcDNA3/sVPI-C3d3, peDNA3/sVP1 and pcDNA3 plasmids respectively once every 4 weeks for 3 times . 14 days after every inoculation venous blood was collected to measure the titer of neutralizing antibody. Three weeks after the last inoculation their spleens were taken out to detect the specific CTL eytotoxie activity. Three mice from each group underwent intraperitoneal injection of 3 LD50 CVB3 and were killed 7 days later, and then the titer of blood viruses was measured. The remaining 8 mice underwent intraperitoneal injection of 5 LD50 CVB3 and the survival status was observed for 21 days. Results The titer of neutralizing antibody in the mouse blood increased along with the time after inoculation. The antibody titer and specific CTL cytotoxic activity 3 days after inoculation of the pcDNA3/sVP1-C3d3 group were 33.6 ± 1.7 and 66. 1% ± 2.9% respectively, both significantly stronger than those of the pcDNA3/sVP1 group [ （28.3 ± 1.7 ） and （52.8% ± 3.3% ） respectively, both P 〈0.05]. After lethal CVB3 challenge, the blood virus titer of the pcDNA3/sVP1-C3d3 group was significantly lower than that of the pcDNA3 group （P 〈0.05 ） , and the survival rate of pcDNA3/sVP1-C3d3 group was 50% , significantly higher than that of the pcDNA3 group （0, P 〈 0. 05 ） , however, not significantly different from that of the pcDNA3/sVP1 group （25% , P 〉 0.05 ）. Conclusion C3d strongly enhances the specific immune response induced by SVP1 gene vaccination.