体内电穿孔增强日本血吸虫核酸疫苗免疫保护作用的研究

Enhancing protective immunity effects of nucleic acid vaccines against Schistosoma japonicum infection through electroporation in vivo

目的探讨体内电穿孔技术增强日本血吸虫核酸疫苗的免疫保护效果。方法分别大量制备质粒pcDNA3.1-SjC23、pcDNA3.1-SjCTPI、pcDNA3.1-(CDR3)6和重组蛋白SjC23-HD、SjCT-PI与NP30。上述3种质粒DNA以等量混合后即为鸡尾酒式DNA疫苗,3种蛋白以等量混合后即为鸡尾酒式蛋白疫苗。70只BALB/c小鼠随机分为A、B、C、D、E5组,每组14只。A组为自然感染组;B组(电脉冲空质粒对照组)每只小鼠分别在第0、3、6周经股四头肌注射100μlpcDNA3.1,每次注射时辅以体内电穿孔;C组(电脉冲空质粒+混合蛋白对照组)空质粒免疫及体内电穿孔同B组,但于第9周每鼠经背部皮下多点注射100μl混合蛋白疫苗+100μl福氏完全佐剂(FCA);D组(电脉冲混合DNA组)每只小鼠分别在第0、3、6周经股四头肌注射100μl混合DNA疫苗,每次免疫时辅以体内电穿孔;E组(电脉冲混合DNA+混合蛋白组)混合DNA免疫及体内电穿孔同D组,但于第9周每鼠经背部皮下多点注射100μl混合蛋白疫苗+100μlFCA。DNA免疫组末次免疫后4周,蛋白加强组末次免疫后2周,所有小鼠同时经腹部皮肤感染(40±1)条尾蚴。攻击感染后42d剖杀小鼠,计数成虫及肝脏虫卵数。首次免疫前2d及感染前2d分别经尾静脉采血,分离血清检测IgG抗体水平、抗体亚类IgG1及IgG2a,并取小鼠脾脏制备单个脾细胞,检测细胞因子IL-2、IL-4、IFN-γ的水平。结果C、D组和E组的减虫率分别为18.09%、45.00%和57.09%,D组和E组的减虫率均显著高于C组(P均<0.01),且E组的减虫率高于D组(P<0.05);C、D组和E组的减卵率分别为12.49%、50.88%和59.26%,D组和E组的减卵率均显著高于C组(P均<0.01),且E组的减卵率高于D组(P<0.05)。C、D、E3组小鼠血清都检测到特异性IgG抗体,抗体亚类IgG2a/IgG1比值分别为0.394、3.518、0.914。D、E2组小鼠IL-2、IFN-γ含量较对照组均有明显升高,IL-4则无明显差异。结论体内电穿孔技术可显著提高日本血吸虫核酸疫苗的免疫保护作用。

Objective To enhance the protective immunity effects of nucleic acid vaccines against Schistosorna japonicum infection by electroporation （EP） in vivo in infected BALB/c mice. Methods Plasmids and proteins for inmmunization were prepared and diluted in no bacterial saline solution tofinal concentration of 1.5 mg/ml, pcDNA3.1-SjC23, pcDNA3.1-SjCTPI, pcDNA3.1-（ CDR3）6 plasmid DNAs were mixed by equal volume to form the cocktail DNA vaccine, and also mixed with recombinant proteins SjC23-HD, SjCTPI, and NP30 by equal volume to form the cocktail protein vaccine. Seventy female BALB/c mice of 4-5 weeks old were randomly divided into 5 groups （A, B, C, D, E）. In Group A （control group）, each mouse was immunized with 100μl saline solution by intramuscular （i. m. ） ; in Group B （pcDNA3.1/EP control group）, each mouse was immunized （i. m. ） with 100μl pcDNA3.1 followed by EP in vivo for three times at week 0, 3, 6; in Group C （pcDNA3.1/EP plus cocktail protein vaccine group）, each mouse was immunized （i. m. ） with 100μl pcDNA3.1 followed by EP for three times at week 0, 3, 6 and boosted with 100 μl cocktail protein vaccine plus 100μl FCA by subcutaneous at week 9; in Group D （cocktail DNA vaccine/EP group）, each mouse was immunized （i. m. ） with 100μl cocktail DNA vaccine followed by EP for three times at week 0, 3, 6; in Group E （cocktail DNA vaccine/EP plus cocktail protein vaccine group）, each mouse was immunized （i. m. ） with 100 μl cocktail DNA vaccine followed by EP for three times at week 0, 3, 6 and boosted with 100μl cocktail protein vaccine plus 100μl FCA by subcutaneous at week 9. Four weeks after the last DNA immunization or two weeks after protein boosting, all the mice were challenged with （40± 1） cercariae of Schistosoma japonicum by abdominal skin penetra- tion. Forty-two days post-challenge, the mice were sacrificed and perfused, and the numbers of recovered worms and eggs in liver were counted. The blood was collected from the tail veins of all the mice two days before the first immunization and challenge, respectively, the serum was prepared for detection of IgG, IgG1 and IgG2a. Two days before the challenge, the spleen cells of two mice from each group were cultured and stimulated with ConA and soluble egg antigen （SEA）, and the supernatant was collected for detection of IL-2, IL-4 and IFN-7 by flow cytometre. Results The worm reduction rates in Group C, D and E were 18.09%, 45.00% and 57.09%, respectively, compared with the control group. The worm reduction rates in Group D and E were significantly higher than that in Group C （P〈0. 01）, also in Group E it was significantly higher than that in Group D （P〈 0.05）. The liver egg reduction rates in Group C, D and E were 12.49 %, 50.88% and 59.26 %, respectively, compared with the control group. The egg reduction rates in Group D and E were significantly higher than that in Group C （P〈0.01）, also in Group E it was higher than that in Group D （P〈0.05）. ELISA results showed that the mice in Group C, D and E produced specific IgG, while the mice in the control group （Group A and B） did not. The mice in Group C, D and E also produced IgG1 and IgG2a antibody isotypes, with the ratios of IgG2a/IgG1 0. 394, 3. 518 and 0. 914, respectively. In comparison with the control group （Group A and B）, the levels of IL-2 and IFN-7 of the mice in Group D and E significantly augmented. Conclusion The protective immunity effects of nucleic acid vaccines against Schistosoma japonicum infection are significantly improved by EP in vivo.