Ag85B与MPT64 DNA疫苗联合免疫对鼠结核分枝杆菌感染的保护作用

目的　研究Ag85B与MPT6 4DNA疫苗联合免疫对鼠结核分枝杆菌感染的免疫保护效果。方法　C5 7BL/ 6小鼠 5 4只 ,采用随机数字表法随机分为 6组 ,分别用磷酸盐缓冲液 (PBS)、pcDNA3 1( )、卡介苗 (BCG)、pcDNA/Ag85B、pcDNA/MPT6 4、pcDNA/Ag85B pcDNA/MPT6 4经肌肉注射法免疫小鼠 ,0、3、6周各 1次 ,即PBS组、pcDNA3 1组、BCG组、pcDNA/Ag85B组、pcDNA/MPT6 4组及pcDNA/Ag85B pcDNA/MPT6 4组。BCG组只在 0周予皮内注射卡介苗 1次。末次免疫后第 5周用 1×10 6CFU的H3 7Rv经尾静脉实施攻击 ,攻击后 6周处死部分小鼠 ,用酶联免疫吸附测定 (ELISA)法检测血清总IgG、纯化蛋白衍生物 (PPD)特异性脾淋巴细胞干扰素γ(IFN γ)和白细胞介素 4 (IL 4 )分泌水平 ,四甲基偶氮唑盐 (MTT)法测定特异性脾淋巴细胞增殖 ;作脾、肺组织荷菌量和病理学检查 ,并观察小鼠存活时间。结果　PBS组肺和脾器官荷菌量分别为lg-1(7 85 4± 0 0 0 3)CFU/g和lg-1(7 190±0 0 16 )CFU/g、pcDNA3.1组分别为lg-1(7 70 0± 0 0 16 )CFU/g和lg-1(7 0 72± 0 0 6 8)CFU/g、BCG组分别为lg-1(6 4 4 9± 0 0 0 2 )CFU/g和lg-1(5 4 36± 0 0 4 2 )CFU/g、pcDNA/Ag85B组分别为lg-1(7 370± 0 0 0 2 )CFU/g和lg-1(6 4 2 96± 0 0 0 9)C

A study of the protective effect of the DNA vaccine encoding tubercle antigen 85B with MPT64 in mice challenged with Mycobacterium tuberculosis

OBJECTIVE: To explore the protective effect of the DNA vaccine encoding Mycobacterium tuberculosis Ag85B with MPT64 in mice infected with Mycobacterium tuberculosis. METHODS: Fifty four C57BL/6 mice were randomized into six groups and subjected to the following treatments respectively: intramuscularly immunized with PBS, pcDNA3.1, BCG, pcDNA/Ag85B, pcDNA/MPT64 and pcDNA/Ag85B + pcDNA/MPT64 on three occasions at 3-week intervals. The BCG group received a single subcutaneous injection of 1 x 10(6) CFU BCG. The mice were challenged with 10(6) CFU H(37)Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed, and the number of CFU in organs and histopathologic changes were determined. The antibody level, IFN-gamma, IL-4 and the survival time in all of the mice were evaluated. RESULTS: The number of bacterial colonies in the lungs and spleens were lg(-1) (7.854 +/- 0.003) CFU/g and lg(-1) (7.190 +/- 0.016) CFU/g in PBS group, lg(-1) (7.700 +/- 0.016) CFU/g and lg(-1) (7.072 +/- 0.068) CFU/g in pcDNA3.1 group, lg(-1) (6.449 +/- 0.002) CFU/g and lg(-1) (5.436 +/- 0.042) CFU/g in BCG group, lg(-1) (7.370 +/- 0.002) CFU/g and lg(-1) (6.430 +/- 0.009) CFU/g in pcDNA/Ag85B group, lg(-1) (7.547 +/- 0.003) CFU/g and lg(-1) (6.784 +/- 0.002) CFU/g in pcDNA/MPT64 group, and lg(-1) (6.918 +/- 0.002) CFU/g and lg(-1) (6.079 +/- 0.004) CFU/g in pcDNA/Ag85B + pcDNA/MPT64 group respectively, which showed significant decrease at 6th week postchallenge in all the vaccinated groups (P < 0.05), especially in BCG group (P < 0.01). Antibody titer of pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/Ag85B group was higher than that of the other groups (P < 0.05). The level of IFN-gamma produced by spleen lymphocytes and spleen lymphocyte proliferation from BCG group, pcDNA/Ag85B + pcDNA/MPT64 group was higher than that of the other groups (P < 0.05). No IL-4 was detected in all groups. The pulmonary histopathological changes were observed 6 weeks later following challenge with Mycobacterium tuberculosis H(37)Rv. In PBS and pcDNA3.1 groups, the lesion was characterized by inflammatory infiltration and lung tissue necrosis, in BCG group by granulomas and numerous macrophages, lymphocytes and a few epithelioid cells. The lesion in pcDNA/Ag85B group was characterized by serofibrous inflammatory infiltration and a few macrophages, in pcDNA/Ag85B + pcDNA/MPT64 group, by granulomas, numerous macrophages and lymphocytes. The lesion in spleen was different from the lung and characterized by proliferative lymphocytes and inflammatory infiltration. The results in spleen were similar to those in the lung. The survival time of BCG vaccinated mice after challenge with Mycobacterium tuberculosis H(37)Rv was longer than that of the other groups. The survival time of pcDNA/Ag85B + pcDNA/MPT64 group was longer than that of other DNA vaccine groups. CONCLUSION: The protective effect of BCG was more significant than the other groups, while the effect of pcDNA/Ag85B + pcDNA/MPT64 was better than other DNA vaccines.