Ag85B-MPT64融合基因疫苗对鼠结核分枝杆菌感染的保护作用

THE ESTIMATION OF PROTECTIVE EFFICACY OF THE FUSION GENE VACCINE ENCODING TUBERCLE ANTIGEN 85B AND MPT64 IN MICE CHALLENGED WITH MYCOBACTERIUM TUBERCULOSIS

目的 研究Ag85B MPT6 4 (AM)融合基因疫苗对鼠结核分枝杆菌感染的保护效果。方法 C5 7BL/ 6小鼠 6 3只 ,随机分为磷酸盐缓冲液 (PBS)、空质粒、卡介苗 (BCG)、pcDNA/Ag85B、pcDNA/MPT6 4、pcDNA/Ag85B +pcDNA/MPT6 4、pcDNA/AM组 ,采用肌肉注射法免疫小鼠 ,0、3、6周各 1次 ,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H37Rv经尾静脉注射实施攻击 ,攻击后 6周处死部分小鼠 ,测血清总IgG ,特异性脾淋巴细胞增殖、IFN γ及IL 4分泌水平 ;观测脾、肺组织荷菌量和病理学检查以及剩余小鼠的存活时间。结果 AM基因疫苗组诱导的特异性的IgG、脾淋巴细胞增殖和IFN γ的分泌以及肺、脾组织荷菌量、病理学改变和小鼠存活时间明显优于其他DNA疫苗单独免疫组。结论 AMDNA疫苗在抗结核分枝杆菌感染过程中具有明显的免疫保护作用。

OBJECTIVE TO EVALUATE THE PROTECTIVE EFFICACY OF THE FUSION DNA VACCINE(AM) ENCODING TUBERCLE AG85B AND MPT64 IN MICE INFECTED WITH MYCOBACTERIUM TUBERCULOSIS. METHODS C57BL/6 MICE WERE INTRAMUSCULARLY IMMUNIZED WITH THE DNA VACCINES. THE MICE WERE CHALLENGED WITH 106 CFU H37RV 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 RESPECTIVELY, AND THE NUMBER OF CFU IN ORGANS AND HISTOPATHOLOGIC CHANGES WAS DETERMINED. THE ANTIBODY LEVEL, IFN-Γ,IL-4 AND THE SURVIVAL TIME IN ALL OF THE MICE WERE EVALUATED. RESULTS ANTIBODY TITER OF PCDNA/AG85B+ PCDNA/MPT64 GROUP AND PCDNA/AM GROUP WAS HIGHER THAN THAT OF OTHER GROUPS (P<0.05).THE LEVEL OF IFN-Γ PRODUCED BY SPLEEN LYMPHOCYTES AND SPLEEN LYMPHOCYTE PROLIFERATION FROM BCG GROUP, PCDNA/AG85B ,PCDNA/AG85B+ PCDNA/MPT64 GROUP AND PCDNA/AM GROUP WAS HIGHER THAN THAT OF OTHER GROUPS (P<0.05). NO IL-4 WAS FOUND IN ALL GROUPS. THE NUMBER OF BACTERIAL COLONIES IN THE LUNGS AND SPLEENS WAS SIGNIFICANTLY DECREASED AT 6TH WEEK POSTCHALLENGE IN ALL THE VACCINATED GROUPS (P<0.05), ESPECIALLY IN BCG GROUP (P<0.01). THE PULMONARY HISTOPATHOLOGICAL CHANGES WERE OBSERVED 6 WEEKS LATER FOLLOWING CHALLENGE WITH M. TUBERCULOSIS H37RV. IN PBS AND PCDNA3.1 GROUPS, THE LESION WAS CHARACTERIZED BY SEROPLASTIC 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 GROUPS WAS CHARACTERIZED BY SEROPLASTIC INFLAMMATORY INFILTRATION AND A FEW MACROPHAGES, IN PCDNA/AG85B+PCDNA/MPT64 GROUP AND PCDNA/AM 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 LUNG. THE SURVIVAL TIME OF BCG VACCINATED MICE AFTER CHALLENGE WITH M.TUBERCULOSIS H37RV WAS LONGER THAN THAT OF OTHER GROUPS. THE SURVIVAL TIME OF AM GROUP WAS LONGER THAN THAT OF OTHER DNA VACCINE GROUPS. CONCLUSION THE PCDNA/AM CAN IMPROVE THE PROTECTIVE EFFICACY IN IMMUNIZED MICE AGAINST M.TUBERCULOSIS.