CVB3VP1核酸疫苗与重组腺病毒Ad/VP1联合应用的免疫效果

Immunological effects of combined application of recombinant DNA vaccine and adenovirus vaccine expressing the protein VP1 of coxsackievirus B3 in mice

目的:观察柯萨奇病毒B3(Coxsackievirus group B type 3,CVB3)VP1核酸疫苗pcDNA3/VP1与重组腺病毒Ad/VP1联合应用的免疫效果。方法:大量制备pcDNA3/VP1和Ad/VP1,肌肉注射免疫小鼠。4～6周龄雄性BALB/c小鼠随机分为4组:质粒组,注射pcDNA3/VP1,共3次;腺病毒组,注射Ad/VP1,共2次;质粒/腺病毒组,第1、2次注射pcDNA3/VP1,第3次注射Ad/VP1;PBS对照组。各次注射间隔时间为3周。质粒每次接种100μg/只,腺病毒每次接种4×107pfu/只。用微量中和试验和ELISA法分别检测每次免疫后血清中和抗体和IgG抗体滴度;CCK-8法检测淋巴细胞增殖活性和CTL杀伤活性;用致死量病毒感染后检测血中病毒滴度,并观察小鼠的生存情况。结果:各实验组小鼠的中和抗体和IgG抗体水平均随免疫次数增加而提高,末次免疫后,质粒/腺病毒组血清中和抗体和特异性IgG抗体滴度(31.70±1.41,2425.15±1.86)明显高于质粒组(23.78±1.37,918.96±1.36)和腺病毒组(18.88±1.22,800.00±1.63)(P<0.05);各实验组淋巴细胞增殖活性和CTL杀伤活性高于PBS对照组(P<0.05);以致死量病毒感染后,质粒/腺病毒组血清病毒滴度低于其他组(P<0.05),生存率高于其他组(P<0.05)。结论:核酸疫苗与重组腺病毒联合应用可以明显提高免疫效果。

Objective：To observe the immunological effects of recombinant vaccines against VP1 of coxsackievirus group B3 （CVB3） via immunization with nucleic acid vaccine pcDNA3/VP1 and adenovirus Ad/VP1 in mice.Methods：Plenty of pcDNA3/VP1 and Ad/VP1 were prepared.Male BALB/c mice of 4-6 week old were randomly divided into four groups：plasmid group was injected by pcDNA3/VP1 for three times.Ad/VP1 group was inoculated with Ad/VP1 for two time.Plasmid-Ad/VP1 group received pcDNA3/VP1 in the first and second times and Ad/VP1 in the third times;Control group was injected with PBS.Mice were intramuscularly immunized into thigh at the intervals of three weeks with 100 μg dose of plasmid or 4×107 pfu dose of Ad/VP1.The titers of sera CVB3VP1 neutralizing antibody and IgG antibody were detected by trace neutralization assay and ELISA.Three weeks after the last immunization,the proliferation of spleen lymphocytes and specific CTL cytotoxic activity were tested by CCK-8.Mice in each group were challenged with lethal dose of CVB3,the titers of sera virus were titrated and the protection efficacy against CVB3 was observed.Results：Titers of CVB3-specific neutralizing antibody and IgG antibody in sera of the mice in every experimental groups were growing along with the immune times （P〈0.05）.Plasmid plus Ad/VP1 group produced higher titers of neutralizing antibody （31.70±1.41） and IgG antibody （2425.15±1.86）than those of mice immunized with either pcDNA3/VP1 （23.78±1.37,918.96±1.36）or Ad/VP1 （18.89±1.22,800.00±1.63）（P〈0.05） alone after the third immunization.After challenged with lethal dose of CVB3,the titers of blood virus in plasmid-Ad/VP1-immunized group were significantly lower than in the other groups （P〈0.05）.Survival rates of the mice in plasmid-Ad/VP1-immunized group were significantly higher than in the other groups （P〈0.05）.Conclusion：Combined application of DNA prime-adenovirus vaccine boost regimen effectively induced stronger immune responses than either plasmid or recombinant adenovirus vaccine alone.