不同剂量结核病DNA疫苗及细胞因子联合免疫的研究

The evaluation of protective efficacy of tuberculosis DNA vaccines with different dosage and by codelivery of cytokine plasmid

目的：评价结核分枝杆菌MPT64（简称M64）和ESAT6（简称E6）DNA疫苗不同剂量联合免疫、与细胞因子联合免疫的保护效力，以及Ag85A（简称85A）和Ag5B（简称85B）DNA疫苗的保护效力。方法：将C57BL／6小鼠随机分为14组免疫：①生理盐水；②载体pVAX1 100μg；③卡介苗；④M64100μg＋E6100μg；⑤M6450μg＋E650μg；⑥M6475μg＋E625μg；⑦M6425μg＋E675μg；⑧M6425μg＋E625μg；⑨M64100μg＋IFN-γ 100μg；⑩E6100μg＋IFN-γ100μg；（11）M64100μg＋IL-12 100μg；（12）E6100μg＋IL-12 100μg；（13）85A100μg；逼（14）5B100μg。用ELISA法检测血清抗体，结核分枝杆菌通过尾静脉攻击小鼠，动态观察小鼠体重变化；攻击4周后，取肺、肝和脾观察病理改变、称重量、做菌落计数。结果：不同剂量的M64和E6DNA联合免疫、85A和85BDNA分别免疫均能诱导小鼠产生特异性抗体，但M64或E6与IFN-γ或IL-12质粒DNA共同免疫后特异性抗体水平与对照组无显著性差异；各组血清抗体亚型IgG2a均无显著升高，IgG2b均显著高于IgGI。第2、3A、8、9、11、12组在感染后4周体重明显增加，而第1、14、5、6组体重下降；第3、11、9、7组的肺菌落指数较少。第10、13组肺肉眼未见明显病变；第2～4、7、9～13组肺组织主要为不同程度的增殖性反应，第1、6、8、14组肺组织为增殖性反应与渗出性反应并存，第5组为渗出性反应。结论：DNA免疫的量需100μg才能诱导产生强的保护效力；M64和E6DNA各100μg混合免疫、85ADNA疫苗的保护效力与卡介苗相当；b164和E6DNA与IFN-γ或IL-12质粒DNA共同免疫后，明显增强其保护效力。

Objective： To evaluation the protective efficacy of immunization with different amount of tuberculosis DNA vaccines and codelivery of genes encoding cytokines. Methods：C57BL/6 mice were randomly divided into 14 groups, immunized with （1）the saline, （2）plasmid vector, （3）M. boris BCG, （4） 100 μg M64 and 100 μg E6 DNA, （5）50 μg M64 and 50 μg E6 DNA, （6）75 μg M64 and 25 μg E6 DNA, （7）25 μg M64 and 75 μg FA DNA, （8）25 μg M64 and 25 μg E6 DNA, （9） 100 μg M64 and 100 IFN-γ/DNA, （10） 100 μg E6 and 100 μg IFN-γ/DNA, （ 11 ） 100 μg M64 and 100 μg IL-12 DNA, （12） 100 μg FA and 100 μg IL- 12 DNA, （13）85A DNA 100 μg, （14）85B DNA 100 μg, and then infected by intravenous injection with M. tuberculosis. Antigenspecific antibodies were determined by ELISA. The body weights of mice were weighed each week. The lungs, livers and spleens were taken and observed their pathological change, weighted and performed mycobacterial cultures. Results：The high levels of antigen-specific antibody could be detected in group 4 to 8, 13 and 14. But antigen-specific antibody did not increase in group 9 to 12. IgG2a isotype was not predominant in each group. Their ratio of antigen-specific antibody IgG2b to IgG1 obviously increased. These results suggested that they generated a Thl-biased response. The body weights in group 2, 3,4, 8, 9, 11,12 obviously increased at 4 weeks after infection, while in group 1, 14, 5,6 decreased. The CFUs of lungs in group 3, 11,9,7 were lower. No lesions could be observed in hngs in group 10 and 13 by eyes. The different degree of hyperplasia reaction in lung tissues could be observed in group 2 to 4, 7, and 9 to 13. The hyperplasia and inflammatory infiltration reaction in lung tissues could he observed in group 1,6, 8, 14. While only inflammatory infihration reaction in lung tissues could be observed in group 5. Conclusion ：The immunization with 100 μg of DNA vaccine could induce strong protection. The immunization with 100 μg of M64 and FA DNA vaccines or 85A DNA vaccine could provide similar protective efficacy as live BCG. The codel/very of genes encoding cytokines IFN-γ or IL-12 could increase the effectiveness of DNA vaccines, especially IFN-γ.