结核亚单位疫苗AEC/BC-C02诱导小鼠长期的抗原特异性细胞应答

中国防痨杂志 ›› 2013, Vol. 35 ›› Issue (1): 32-36.

结核亚单位疫苗AEC/BC-C02诱导小鼠长期的抗原特异性细胞应答

卢锦标杨蕾付丽丽陈保文都伟欣王国治徐苗

100050 北京，中国食品药品检定研究院结核病疫苗室（卢锦标、杨蕾、陈保文、都伟欣、王国治、徐苗）；温州医学院环境与公共卫生学院（付丽丽）

收稿日期:2012-11-20 出版日期:2013-01-10 发布日期:2013-04-03
通信作者: 徐苗 E-mail:xumiaobj@126.com
基金资助:
“十二五”国家科技重大专项（2012ZX10004701）

Mycobacterium tuberculosis subunit vaccine AEC/BC-C02 induced long term antigen-specific cellular immune response in mice

LU Jin-biao, YANG Lei, FU Li-li，CHEN Bao-wen, DU Wei-xin，WANG Guo-zhi, XU Miao

Division of Tuberculosis Vaccines, National Institutes for Food and Drug Control, Beijing 100050, China

Received:2012-11-20 Online:2013-01-10 Published:2013-04-03
Contact: XU Miao E-mail:xumiaobj@126.com

摘要/Abstract

摘要： 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-γ SFC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。

关键词: 疫苗, 亚单位, 免疫, 细胞, 抗原, 细菌, 细菌蛋白质类, 重组融合蛋白质类, 小鼠

Abstract: Objective To evaluate dynamically cellular immune response of Mycobacterium tuberculosis subunit vaccine AEC/BC-C02 in mice. Methods Forty-eight female BALB/c mice (6-8 weeks old) were randomly divided into two groups. One group was immunized intramuscularly 3 times with AEC/BC-C02 at an interval of 10 days, and the other with PBS as the control. At 1, 2, 4 and 8 weeks after the last vaccination, spleen lymphocytes were isolated (6 mice per group) to measure the frequency of antigen-specific IFN-γ secreting T cell by ELISPOT. Meanwhile, antigen-specific IFN-γ level was detected by ELISA at 2, 4 and 8 weeks, and lymphoproliferation response was measured by cell counting kit-8 assay at 4 and 8 weeks. Results (1) At 1, 2, 4 and 8 weeks after the last vaccination, the spot forming cells (SFC) of Ag85B-specific IFN-γ in vaccine group were respectively 168.8±103.5, 205.2±51.0, 206.8±65.3 and 160.0±67.9, and significantly higher than that in PBS group (8.9±6.0, t=3.779, P<0.01; 16.1±18.8, t=8.525, P<0.01; 9.3±4.9, t=7.424, P<0.01 and 7.7±6.6, t=5.473, P<0.01, respectively). ESAT6/CFP10 (EC)-specific IFN-γ SFC in vaccine group were respectively 45.1±18.6, 75.6±39.3, 86.2±50.4 and 54.3±26.3, and significantly higher than that in PBS group(4.5±3.5, t=5.258, P<0.01; 11.7±10.5, t=3.850, P<0.01; 3.8±5.8, t=3.977, P<0.01 and 5.2±4.3, t=4.521, P<0.01, respectively). (2) At 2, 4 and 8 weeks after the last vaccination, IFN-γ release induced by Ag85B peptide pool in vaccine group were (1.27±0.13)ng/ml, (1.76±0.55)ng/ml and (1.44±0.44)ng/ml, respectively, and that induced by EC peptide pool were (0.81±0.33)ng/ml, (0.81±0.69)ng/ml and (0.54±0.29)ng/ml, respectively. The difference between them was statistically significant (t=3.008, P<0.05; t=2.631, P<0.05 and t=10.02, P<0.01, respectively). (3) At 4 and 8 weeks after the last vaccination, stimulation indexes (SI) of spleen lymphocytes induced by Ag85B peptide pool were respectively 1.756±0.339 and 1.936±0.287 in vaccine group and signi-ficantly higher than PBS group (1.287±0.0581, t=3.030, P<0.05 and 1.382±0.114, t=4.387, P<0.01, respectively). SI of spleen lymphocytes induced by EC peptide pool in vaccine group were respectively 1.599±0.154 and 1.581±0.156 and significantly higher than PBS group (1.380±0.126, t=2.540, P<0.05 and 1.314±0.170, t=2.844, P<0.05, respectively). Conclusion Mycobacterium tuberculosis subunit vaccine could induce long term and stable antigen-specific memory T cells in mice, which provided the basis for the late-stage study of its protective immunity against Mycobacterium tuberculosis.

Key words: Vaccines, subunit, Immunity, cellular, Antigens, bacterial, Bacterial proteins, Recombinant fusion proteins, Mice

卢锦标杨蕾付丽丽陈保文都伟欣王国治徐苗. 结核亚单位疫苗AEC/BC-C02诱导小鼠长期的抗原特异性细胞应答[J]. 中国防痨杂志, 2013, 35(1): 32-36.

LU Jin-biao, YANG Lei, FU Li-li，CHEN Bao-wen, DU Wei-xin，WANG Guo-zhi, XU Miao. Mycobacterium tuberculosis subunit vaccine AEC/BC-C02 induced long term antigen-specific cellular immune response in mice[J]. Chinese Journal of Antituberculosis, 2013, 35(1): 32-36.

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