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目的动态评价结核亚单位候选疫苗AEC/BC-C02在小鼠中的细胞免疫应答水平。方法6～8周龄SPF级BALB/c雌鼠48只,按数字表法随机分成两组,一组免疫AEC/BC-C02,另一组免疫PBS。末次免疫后第1、2、4、8周分别取两组小鼠(6只/组)分离脾淋巴细胞,ELISPOT检测分泌抗原特异性IFN-γ的T细胞频率;在第2、4、8周ELISA检测抗原特异性IFN-γ的分泌量;在第4、8周,Cell Counting Kit-8(CCK-8)法检测脾淋巴细胞增殖。结果 (1)末次免疫后第1、2、4、8周,对疫苗组小鼠脾淋巴细胞,Ag85B特异性的IFN-γ斑点形成细胞(spot forming cells,SFC)分别为168.8±103.5、205.2±51.0、206.8±65.3和160.0±67.9,与PBS对照组的8.9±6.0、16.1±18.8、9.3±4.9和7.7±6.6比较,差异均有统计学意义(成组t检验,t值分别为3.779、8.525、7.424、5.473;P值均<0.01);EC特异性的IFN-γSFC分别为45.1±18.6、75.6±39.3、86.2±50.4和54.3±26.3,与PBS对照组的4.5±3.5、11.7±10.5、3.8±5.8和5.2±4.3比较,差异均有统计学意义(成组t检验,t值分别为5.258、3.850、3.977、4.521;P值均<0.01)。(2)末次免疫后第2、4、8周,对疫苗组小鼠脾淋巴细胞,Ag85B多肽刺激的IFN-γ分泌量分别是(1.27±0.13)ng/ml,(1.76±0.55)ng/ml和(1.44±0.44)ng/ml;EC多肽刺激的IFN-γ分泌量分别是(0.81±0.33)ng/ml,(0.81±0.69)ng/ml和(0.54±0.29)ng/ml,两者间差异有统计学意义(配对t检验,分别为:t=3.008,P<0.05;t=2.631,P<0.05;t=10.02,P<0.01)。(3)末次免疫后第4、8周,Ag85B多肽对疫苗组小鼠脾淋巴细胞的刺激指数(SI)分别为1.756±0.339和1.936±0.287,均分别高于PBS组的1.287±0.0581和1.382±0.114,差异有统计学意义(成组t检验,分别为:t=3.030,P<0.05;t=4.387,P<0.01);EC多肽对疫苗组SI分别为1.599±0.154和1.581±0.156,均分别高于PBS组的1.380±0.126和1.314±0.170,差异有统计学意义(成组t检验,t值分别为2.540、2.844;P值均<0.05)。结论新型结核亚单位疫苗AEC/BC-C02免疫小鼠可诱导长期稳定存在的抗原特异性记忆T细胞,为后期抗Mtb保护力研究提供药理学基础。
Objective To dynamically evaluate the level of cellular immune response in mice by the candidate subunit vaccine AEC / BC-C02. Methods 48 BALB / c females aged from 6 to 8 weeks old were randomly divided into two groups according to the digital table. One group was immunized with AEC / BC-C02 and the other group was immunized with PBS. Spleen lymphocytes were isolated from two groups of mice (6 mice / group) at 1, 2, 4 and 8 weeks after the last immunization, and the frequency of T cells secreting antigen-specific IFN-γ was detected by ELISPOT. Week ELISA to detect the secretion of antigen-specific IFN-γ; at 4 and 8 weeks, Cell Counting Kit-8 (CCK-8) method to detect splenic lymphocyte proliferation. Results (1) At the 1st, 2nd, 4th, and 8th week after the last immunization, the specific formation of Ag85B IFN-γ in the spleen lymphocytes of mice in the vaccine group was 168.8 ± 103.5 and 205.2 ± 51.0,206.8 ± 65.3 and 160.0 ± 67.9, respectively, which were significantly different from those of the PBS control group (8.9 ± 6.0, 16.1 ± 18.8, 9.3 ± 4.9 and 7.7 ± 6.6, respectively) (group t test, t = 3.779 , 8.525,7.424,5.473, P <0.01). The EC-specific IFN-γSFCs were 45.1 ± 18.6, 75.6 ± 39.3, 86.2 ± 50.4 and 54.3 ± 26.3, respectively, 10.5, 3.8 ± 5.8 and 5.2 ± 4.3 respectively. The differences were statistically significant (group t test, t values were 5.258, 3.850, 3.977, 4.521, P <0.01 respectively). (2) At the second, fourth, eighth and eighth weeks after the last immunization, the amounts of IFN-γ secreted by splenic lymphocytes and Ag85B in vaccine group were (1.27 ± 0.13) ng / ml and (1.76 ± 0.55) (0.81 ± 0.33) ng / ml, (0.81 ± 0.69) ng / ml and (0.54 ± 0.29) ng / ml, respectively. The levels of IFN- (Paired t test, t = 3.008, P <0.05; t = 2.631, P <0.05; t = 10.02, P <0.01). (3) At the 4th and 8th week after the last immunization, the spleen lymphocyte stimulation index (SI) of Ag85B polypeptide in vaccine group was 1.756 ± 0.339 and 1.936 ± 0.287, respectively, which were higher than that of PBS group (1.287 ± 0.0581 and 1.382 ± 0.114, the difference was statistically significant (group t test, t = 3.030, P <0.05; t = 4.387, P <0.01) (1.380 ± 0.126 and 1.314 ± 0.170, respectively) in PBS group (t-test, t = 2.540 and 2.844 respectively; all P <0.05). Conclusion AEC / BC-C02 immunized mice could induce long-term stable existence of antigen-specific memory T cells, providing a pharmacological basis for the study of anti-Mtb protection in the late stage.