日本血吸虫鸡尾酒式DNA疫苗与蛋白疫苗联合应用增强免疫保护作用的研究

Enhancing protective immunity effects of the vaccine against Schistosoma japonicum infection through priming with cocktail DNA vaccines and boosting with cocktail protein vaccines

目的探讨日本血吸虫鸡尾酒式DNA疫苗与蛋白疫苗联合应用以增强免疫保护作用的效果。方法分别大量制备质粒DNA:pcDNA3.1-SjC23、pcDNA3.1-SjCTPI、pcDNA3.1-(CDR3)6和重组蛋白SjC23-HD、SjCTPI、NP30。pcDNA3.1-SjC23、pcDNA3.1-SjCTPI、pcDNA3.1-(CDR3)6等量混合后即为鸡尾酒式的混合DNA疫苗,重组蛋白SjC23-HD、SjCTPI、NP30等量混合后即为鸡尾酒式的混合蛋白疫苗。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(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;C组(空质粒+混合蛋白对照组)每只小鼠分别在第0、3、6周经股四头肌注射100μlpcDNA3.1,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(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;E组(混合DNA+混合蛋白组)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,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17.70%、32.88%和45.35%,D组和E组的减虫率均显著高于C组(P均<0.01),且E组的减虫率显著高于D组(P<0.01);C、D组和E组的减卵率分别为9.39%、36.20%和48.54%,D组和E组的减卵率均显著高于C组(P均<0.01),且E组的减虫率也显著高于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0.525、1.829、0.712。D、E两组小鼠IL-2、IFN-γ含量较空质粒对照组均有明显升高,IL-4则无明显差异。结论混合DNA疫苗和混合蛋白疫苗联合应用具有一定的正协同作用,混合蛋白疫苗可显著增强混合DNA疫苗的免疫保护作用。

Objective To enhance the protective immunity effects against Schistosoma japonicum infection by priming with cocktail DNA vaccines and boosting with cocktail protein vaccines in infected BALB/c mice. Methods Plasmids and proteins for immunization were prepared and diluted in no bacterial saline solution to final concentration of 1.5 mg/ml, and mixed with pcDNA3.1-SjC23, pcDNA3. 1-SjCTPI, pcDNA3.1-（CDR3）6 plasmid DNAs 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 100 μl saline solution by intramuscular （i. m. ） ; in Group B （pcDNA3. 1 control group）, each mouse was immunized （i. m. ） with 100 μl pcDNA3.1 for three times at week 0, 3, 6; in Group C （pcDNA3. 1 and cocktail protein group）, each mouse was immunized （i. m. ） with 100 μl pcDNA3.1 for three times at week 0, 3, 6 and immunized with 100 μl mixed protein vaccines plus 100 μl FCA by subcutaneous at week 9; in Group D （cocktail DNA vaccines group）, each mouse was immunized （i. m. ） with 100 μl mixed DNA vaccines for three times at week 0, 3, 6; in Group E （cocktail DNA vaccines plus cocktail proteins）, each mouse was immunized （i. m. ） with 100 μl mixed DNA vaccines for three times at week 0, 3, 6 and immunized with 100 μl mixed protein vaccines plus 100 μl 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 penetration at the same time. 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-γ. Results The worm reduction rates in Group C, D and E were 17. 70%, 32. 88% and 45. 35%, 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 was significantly higher than that in Group D （P〈0. 01）. The egg reduction rates in Group C, D and E were 9.39%, 36.20% and 48. 54%, 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 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 to cocktail protein vaccines, 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. 525, 1. 829 and 0. 712, respectively. In comparison with the control group （Group A and B）, the levels of IL-2 and IFN-γ of the mice in Group D and E were obviously augmented. Conclusions Priming with cocktail DNA vaccines and boosting with cocktail protein vaccines may enhance the protective immunity effects against Schistosoma japonicum infection. It is a new strategy in vaccination for enhancing the protective effect.