IL-12联合结核杆菌DNA疫苗初免-BCG加强免疫的免疫效果观察

Immunogenicity of Interleukin 12 and DNA Vaccine Prime-BCG Boost Against Mycobacterium Tuberculosis

目的研究并比较IL-12联合结核分枝杆菌(TB)DNA(Ag85A和ESAT-6)疫苗初免-BCG加强免疫的应答效果。方法将小鼠随机分成PBS阴性对照组和4组免疫组:BCG组、DNA(Ag85A和ESAT-6)初免-BCG异源加强组、DNA+IL-12初免-BCG异源加强组和DNA初免-BCG+IL-12异源加强组。末次免疫后4、6、8周分别测定血清总IgG水平,同时分离小鼠脾细胞,进行淋巴细胞增殖实验(流式细胞仪检测),测定脾细胞培养上清中IFN-γ分泌水平,并检测小鼠脾脏淋巴细胞表型。结果4组免疫组体外经TB纯蛋白衍生物(TB-PPD)刺激后均检测到特异性IgG抗体产生,且抗体水平在末次免疫后4～8周逐渐增加,4组平均效价为1∶80,1∶120、1∶160、1∶160,各组抗体水平增加无明显差异(P>0.05);小鼠脾细胞体外经TB-PPD刺激后,4组免疫组均能产生特异性淋巴细胞增殖并诱生较强的IFN-γ反应,且DNA+IL-12/BCG和DNA/BCG+IL-12组特异性淋巴细胞增殖活性和IFN-γ分泌水平均明显强于BCG,DNA/BCG组(P<0.05),但DNA+IL-12/BCG和DNA/BCG+IL-12组间差异不明显;4组免疫组小鼠CD4+、CD8+T淋巴细胞较PBS组有较大升高(P<0.05),且DNA+IL-12/BCG和DNA/BCG+IL-12组CD4+、CD8+T淋巴细胞百分比大于BCG以及DNA/BCG组(P<0.05)。结论IL-12联合DNA疫苗初免-BCG加强的免疫策略较BCG免疫以及单纯的DNA疫苗初免-BCG加强免疫能在小鼠体内诱导更强的特异性细胞免疫反应,产生高水平的IFN-γ。

Objective To investigate the immunogenicity of vaccine strategies about human interleukin 12 associated with combined DNA（Ag85A and ESAT-6）prime-BCG boost.Methods BALB/c mice were divided into PBS negative control and 4 immunity groups：BCG group,DNA/BCG group,DNA＋IL-12/BCG group and DNA/BCG＋IL-12 group.All mice received three immunizations at 2-week interval.Specific IgG antibody in serum of mice was determined with indirect ELISA in 4,6,8 weeks respectively after final vaccination.The splenic lymphocytes of mice were separated and stimulated with PPD to measure their proliferation by flow cytometry,and to evaluate the production of interferon-γ（IFN-γ）in cell suspensions of spleen cells by ELISA.The levels of CD4^＋ and CD8^＋ T-cell on surface of spleens lymphocyte were determined by flow cytometry.Results PPD could stimulate specific IgG responses in 4 immunity groups,and the average valences of 4 groups are 1∶80,1∶120,1∶160,1∶160;the splenic lymphocyte proliferation reactions and IFN-γproduction weredetectable in 4 immunity groups,and the most significant response occurred in 12 weeks.DNA＋IL-12/BCG group and DNA/BCG＋IL-12 group induced higher production than BCG group and DNA/BCG group（P〈0.05）,and the effects between DNA＋IL-12/BCG group and DNA/BCG＋IL-12 group had little difference.The numbers of CD4^＋ and CD8^＋ T-cell in 4 immunity groups were much higher than PBS group（P〈0.05）,and DNA＋IL-12/BCG group and DNA/BCG＋IL-12 group were detected much more CD4^＋ and CD8^＋ T-cell than BCG group and DNA/BCG group（P〈0.05）.The level of T-cell between DNA＋IL-12/BCG group and DNA/BCG＋IL-12 group had little difference.Conclusion Interleukin 12 associated with the strategy of priming with the combined DNA vaccines and boosting with attenuated M.bovis vaccine（BCG）could induce much stronger specific cellular immunity compared with simple DNA/BCG or attenuated M.bovis vaccine alone.