结核融合蛋白AMM亚单位疫苗强化BCG初始免疫的免疫效应

The effects of immunity induced by BCG priming and tuberculosis fusion protein AMM vaccine boosting

目的研究结核融合蛋白Ag85B—Mpt64190-198-Mtb8．4（AMM）和佐剂二甲基三十六烷基铵（DDA）、卡介苗多糖核酸（BCG—PSN）构建的亚单位疫苗强化BCG初始免疫的免疫效应。方法将融合蛋白AMM、佐剂DDA和BCG—PSN混合构建AMM亚单位疫苗。实验1组BCG初免后第10周用AMM亚单位疫苗加强免疫小鼠一次；实验2组BCG初免后分别于第8周、第10周用AMM亚单位疫苗加强免疫小鼠一次。同时设立生理盐水及仅BCG免疫两个对照组。BCG初免后第14周、第22周，应用ELISPOT、ELISA检测免疫小鼠的细胞及体液免疫反应。同时在第22周用BCG活菌攻击被免疫小鼠，间隔4周后用流式细胞术和ELISA技术检测T细胞分型及体液免疫反应。结果（1）IFN-γ水平：BCG初免后14周，特异性抗原Ag85B刺激后实验2组分泌IFN-γ的细胞数（135±14）明显高于仅BCG免疫组（19±16），t=10．98，P〈0．01；BCG初免后22周，实验2组（208±11）同样高于仅BCG免疫组（57±18），t=6．43，P〈0．01。（2）体液免疫应答水平：实验2组的IgG1抗体滴度明显高于实验1组，而作为反映Th1型免疫反应指标的IgG2a／IgG1比值，强化免疫两次组低于强化一次组。（3）BCG模拟攻击被免疫小鼠后，CD4^＋CD25^＋调节性T细胞含量：实验1、2组均高于仅BCG免疫组（t1=3．08，t2=3．16，P〈0．05）。结论BCG免疫-AMM亚单位疫苗加强免疫两次能够引起较强的细胞及体液免疫反应，同时激活调节性免疫反应。

Objective To investigate the boosting efficiency of a subunit vaccine consisting of the fusion protein Ag85B-Mpt64190-198-Mtb8.4 （AMM） , dimethyl-dioctyldecyl ammonium bromide （DDA） and BCG polysaccharide nucleic acid （BCG-PSN） on the primed inoculation with BCG. Methods The AMM subunit vaccine was composed of fusion protein AMM, adjuvant DDA and BCG-PSN. The first mouse experimental group was immunized with BCG first, then boosted with the AMM subunit vaccine in the 10th week. The second experimental group was boosted with the AMM subunit vaccine in the 8th week and the lOth week respectively with a two weeks interval after the primed with BCG. Two control groups were treated respectively with physiological saline alone and BCG alone. After the primed inoculation, ELISPOT and ELISA were used for the detection of the cell-mediated and humoral immune response in week 14 and week 22 re- spectively. Furthermore, the immunized mice were challenged with live BCG to mimic tuberculosis infection in the 22nd week after the primed inoculation. Subsequently the T cell typing and humoral response were detected by flow cytometry and ELISA, respectively. Results （ 1 ） The level of secreting IFN-γ ： 14 weeks after the primed inoculation ,with the stimulation of the specific antigen-Ag85B, the number of cells secreting IFN-γin the second experimental group （ 135 ± 14） was more than BCG alone immunized group （ 19 ±16）, t = 10.98, P 〈0.01. In the 22nd week, the number of cells secreting IFN-γ in the second experimental group （208 ± 11 ） was still more than BCG alone group （57±18）, t =6.43, P 〈0.01. （2） The level of humoral immune response： the IgG1 antibody titer in the second experimental group was obviously higher than that in the first experimental group. However, the ratio of IgG2a to IgG1, as the index reflecting the Thl-type immune response, in the experimental group 2 was lower than that in the experimental group 1. （ 3 ） The contents of CD4^＋ CD25 ^4 T cells after challenged with live BCG strain ： the first and the second experimental groups were both higher than the BCG alone group （ t1 = 3.08, t2 = 3.16, P 〈 0.05 ）. Conclusion Boosting the BCG-primed mice with tuberculosis AMM subunit vaccine twice can induce higher level of cell-mediated and hurnoral immune response than BCG alone, which could activate the regulative immune response at the same time.