MF59佐剂增强热灭活卡介苗的免疫原性

doi:10.3969/j.issn.1000-6621.2014.06.007

摘要/Abstract

摘要： 目的研究MF59佐剂［一种由角鲨烯、山梨糖醇三油酸酯(Span85)、吐温80和柠檬酸缓冲液组成的水包油乳剂］对热灭活BCG（hBCG）免疫小鼠诱导免疫应答的影响，验证自制MF59增强hBCG诱导细胞免疫反应的作用。方法 BALB/c（SPF级）小鼠分成A～G组，每组8只，于第0、2、4周皮下注射0.2 ml MF59、低剂量hBCG（0.025 mg/ml）、中剂量hBCG（0.25 mg/ml）、高剂量hBCG（2.5 mg/ml）、MF59+低剂量hBCG、MF59+中剂量hBCG、MF59+高剂量hBCG。末次免疫2周，解剖小鼠。取小鼠脾细胞和腹腔巨噬细胞，并在体外经BCG-PPD刺激培养，ELISA检测培养上清γ干扰素（IFN-γ）、白细胞介素（IL）-1β、IL-2、IL-4、IL-12含量，酶联免疫斑点法（ELISPOT）检测脾细胞分泌IFN-γ、IL-2和IL-4的斑点形成细胞数（SFCs）。所有试验组的平均值比较采用一元方差分析、每两组平均值比较采用最小显著差异法，以P<0.05 为差异有统计学意义。结果 MF59作为高剂量hBCG的佐剂（G组）免疫小鼠，其脾细胞体外经BCG-PPD刺激培养，分泌IFN-γ的SFCs［（151.3±66.6）个］、IL-2的SFCs［（247.8±58.0）个］和IL-4的SFCs［（65.8±24.6）个］显著高于其他组［其他组IFN-γ、IL-2和IL-4的SFCs的最高平均值分别为（30.4±13.0）个、（37.1±10.8）个和（16.4±9.7个）］（分泌IFN-γ的SFCs比较，t=3.2, P=0.007；分泌IL-2的SFCs比较，t=3.6, P=0.003；分泌IL-4的SFCs比较，t=3.0, P=0.01）。MF59与不同剂量hBCG联合免疫小鼠，其腹腔巨噬细胞体外经BCG-PPD刺激培养，培养上清IL-1β水平分别为（663.3±177.5）pg/ml、（813.8±193.4）pg/ml和（742.3±316.1）pg/ml，均显著高于A、B和C组［分别为（104.7±65.8）pg/ml、（82.9±54.8）pg/ml和（66.5±53.5）pg/ml］（E、F和G与A组比较：t=2.9, P=0.01；t=3.5, P=0.004；t=2.5, P=0.031。E、F和G与B组比较：t=3.1, P=0.007；t=3.6, P=0.003；t=2.6, P=0.024。E、F和G与C组比较：t=3.0, P=0.01；t=3.5, P=0.004；t=2.5, P=0.031）。A、C和G组小鼠的腹腔巨噬细胞体外经BCG-PPD刺激培养，培养上清IL-12水平［分别为（36.3±20.5）pg/ml、（94.5±20.6）pg/ml和（128.2±54.6）pg/ml］均显著低于E和F组［（545.7±97.2）pg/ml和（665.5±295.4）pg/ml］（A、C和G组与E组比较：t=－5.1, P=0.000；t=－4.5, P=0.000；t=－3.7, P=0.002。A、C和G组与F组比较：t=－4.4, P=0.001；t=－3.7, P=0.002；t=－2.9, P=0.012）。结论 MF59与高剂量hBCG联合免疫小鼠，可以增强脾细胞体外分泌BCG-PPD特异的IFN-γ、IL-2和IL-4；MF59与低、中剂量hBCG联合免疫小鼠，可以增强腹腔巨噬细胞体外分泌BCG-PPD特异的IL-1β和IL-12。

关键词: 聚山梨醇酯类, 角鲨烯, 卡介苗, 佐剂, 免疫, 细胞因子类

Abstract: Objective To study the effects of MF59 (an oil-in-water emulsion composed of small droplets of squalene surrounded by a monolayer of nonionic detergents (Span85 and Tween 80)) as adjuvant on immune responses to the heat-killed Bacillus Calmette-Guérin (hBCG) to prove MF59 enhancing the cellular immune responses to hBCG. Methods BALB/c mice were divided into seven groups, which were immunized with MF59 (group A), low doses of hBCG (0.025 mg/ml） (group B), middle doses of hBCG (0.25 mg/ml) (group C), high doses of hBCG (2.5 mg/ml) (group D), MF59+ low doses of hBCG (group E), MF59+ middle doses of hBCG (group F) and MF59+ high doses of hBCG (group G) for three times at intervals of two weeks, respectively. The mice were sacrificed two weeks after the last immunization. The splenic lymphocytes and peritoneal macrophages were isolated and cultured with BCG-PPD. Sandwich ELISA and ELISPOT assay were used to detect BCG-PPD-specific cytokines in culture supernatants and the number of IFN-γ, IL-2, IL-4 producing spot forming cells (SFCs), respectively. One-way analysis of variance with least-significant-difference (LSD) comparisons was used to test differences among the levels of cytokines and the number of SFCs. P values of <0.05 were considered statistically significant. Results The number of IFN-γ, IL-2, IL-4 producing SFCs in group G (151.3±66.6, 247.8±58.0, and 65.8±24.6) was higher than that in other groups (the biggest means of IFN-γ, IL-2, IL-4 producing SFCs were (30.4±13.0), (37.1±10.8), and (16.4±9.7), respectively) (t=3.2, P=0.007 for IFN-γ producing SFCs；t=3.6, P=0.003 for IL-2 producing SFCs；t=3.0, P=0.01 for IL-4 producing SFCs). The levels of IL-1β in the supernatants from BCG-PPD-stimulated peritoneal macrophages were higher in groups E (663.3±177.5 pg/ml), F (813.3±193.4 pg/ml), and G (742.3±316.1 pg/ml) than that in groups A (104.7±65.8 pg/ml), B (82.9±54.8 pg/ml) and C (66.5±53.5 pg/ml) (group E vs A, t=2.9, P=0.01; group F vs A, t=3.5, P=0.004; group G vs A, t=2.5, P=0.031; group E vs B, t=3.1, P=0.007; group F vs B, t=3.6, P=0.003; group G vs B, t=2.6, P=0.024; group E vs C, t=3.0, P=0.01；group F vs C, t=3.5, P=0.004；group G vs C, t=2.5, P=0.031). The levels of IL-12 were lower in groups A (36.3±20.5 pg/ml), C (94.5±20.6 pg/ml) and G (128.2±54.6 pg/ml) than that in groups E (545.7±97.2 pg/ml) and F (665.5±295.4 pg/ml) (group A vs E, t=－5.1, P=0.000; group C vs E, t=－4.5, P=0.000; group G vs E, t=－3.7, P=0.002; group A vs F, t=－4.4, P=0.001; group C vs F, t=－3.7, P=0.002; group G vs F, t=－2.9, P=0.012). Conclusion MF59 combined with high dose of hBCG adjuvant can induce BCG-PPD specific IFN-γ, IL-2, and IL-4 secretion from splenic lymphocytes, and MF59 combined with low or middle doses of hBCG adjuvant can increase the levels of IL-1β and IL-12 secreted from the macrophage.

Key words: Polysorbates, Squalene, BCG vaccine, Adjuvants, immunologic, Cytokines

黄香玉，张春青，李军丽，邵进士，何秀云. MF59佐剂增强热灭活卡介苗的免疫原性[J]. 中国防痨杂志, 2014, 36(6): 440-446. doi: 10.3969/j.issn.1000-6621.2014.06.007

HUANG Xiang-yu, ZHANG Chun-qing, LI Jun-li, SHAO Jin-shi, HE Xiu-yun. MF59 adjuvant enhances immunogenicity of heat-killed BCG[J]. Chinese Journal of Antituberculosis, 2014, 36(6): 440-446. doi: 10.3969/j.issn.1000-6621.2014.06.007

参考文献

［1］赵爱华, 卢锦标, 王国治. 我国结核病新疫苗研究策略的探讨. 中华微生物学和免疫学杂志, 2013, 33(12): 885-892.
［2］Stop TB Partnership Working Group on New TB Vaccine. Tuberculosis vaccine candidates 2010. Geneva: World Health Organization,2010. http://www.stoptb.org/.
［3］达泽蛟, 胡丽娜, 王秉翔, 等. 二甲基三十六烷基铵及卡介苗多糖核酸佐剂在结核亚单位疫苗加强BCG免疫中的效应研究. 中华微生物学和免疫学杂志, 2010, 30(6): 555-559.
［4］赵爱华, 李凤祥, 寇丽杰, 等. 卡介菌CpG DNA复合佐剂-02系统(BC-C02)有效性与安全性评价. 中国防痨杂志, 2013, 34(3): 161-167.
［5］O’Hagan DT, Ott GS, De Gregorio E，et al. The mechanism of action of MF59 an innately attractive adjuvant formulation. Vaccine, 2012, 30(29):4341-4348.
［6］Tsai TF. Fluad-MF59-adjuvanted influenza vaccine in older adults. Infect Chemother, 2013, 45(2):159-174.
［7］Ott G, Barchfeld GL, Chernoff D，et al. MF59, design and evaluation of a safe and potent adjuvant for human vaccines. Pharm Biotechnol, 1995, 6:277-296.
［8］Vono M, Taccone M, Caccin P，et al. The adjuvant MF59 induces ATP release from muscle that potentiates response to vaccination. Proc Natl Acad Sci U S A, 2013, 110(52):21095-21100.
［9］Seubert A, Monaci E, Pizza M，et al. The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells. J Immunol, 2008, 180(8):5402-5412.
［10］O′Hagan DT, Ott GS, Nest GV, et al. The history of MF59 adjuvant: a phoenix that arose from the ashes. Expert Rev Vaccines, 2013, 12(1):13-30.
［11］Parkash O. Vaccine against tuberculosis: a view. J Med Microbiol，2014.
［12］Haile M, Schroder U, Hamasur B，et al. Immunization with heat-killed Mycobacterium bovis bacilli Calmette-Guerin (BCG) in Eurocine L3 adjuvant protects against tuberculosis. Vaccine, 2004, 22(11/12):1498-1508.
［13］Kakeda M, Yamanaka K, Kitagawa H，et al. Heat-killed bacillus Calmette-Guérin and Mycobacterium kansasii antigen 85B combined vaccination ameliorates dermatitis in a mouse model of atopic dermatitis by inducing regulatory T cells. Br J Dermatol, 2012, 166(5):953-963.
［14］Dey AK, Burke B, Sun Y，et al. Use of a polyanionic carbomer, Carbopol971P, in combination with MF59, improves antibody responses to HIV-1 envelope glycoprotein. Vaccine，2012，30(17):2749-2759.
［15］Griffiths PD, Stanton A, McCarrell E，et al. Cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant in transplant reci-pients: a phase 2 randomised placebo-controlled trial. Lancet, 2011, 377(9773):1256-1263.
［16］Baudner BC, Ronconi V, Casini D，et al. MF59 emulsion is an effective delivery system for a synthetic TLR4 agonist (E6020). Pharm Res, 2009, 26(6):1477-1485.
［17］Burke B, Gómez-Román VR, Lian Y, et al. Neutralizing antibody responses to subtype B and C adjuvanted HIV envelope protein vaccination in rabbits. Virology, 2009, 387(1):147-156.
［18］Yang M, Yan Y, Fang M，et al. MF59 formulated with CpG ODN as a potent adjuvant of recombinant HSP65-MUC1 for inducing anti-MUC1+ tumor immunity in mice. Int Immunopharmacol, 2012, 13(4):408-416.
［19］李登瑞, 杨永辉, 孙巍. 结核病的先天性免疫与TLR2/4/9的关系. 中国防痨杂志, 2011, 33(3): 173-178.
［20］Chee CB, Gan SH, Khinmar KW，et al. Comparison of sensitivities of two commercial gamma interferon release assays for pulmonary tuberculosis. J Clin Microbiol, 2008, 46(6):1935-1940.
［21］李传友, 邢爱英, 李亮, 等. 结核分支杆菌感染巨噬细胞后氮氧化物的产生及细胞因子表达的研究. 中华结核和呼吸杂志, 2003，26（4）：214-217.
［22］徐苗, 陈保文, 沈小兵, 等. 耻垢分枝杆菌疫苗对小鼠细胞因子产生及Th1/Th2应答的影响. 中华结核和呼吸杂志, 2005, 28(11):781-784.

[1]	赵爱华, 康万里, 王国治, 高正伦, 都伟欣, 卢锦标, 沈小兵, 苏城, 徐苗, 郑素华. 重组结核分枝杆菌11kDa蛋白鉴别潜伏性结核感染与卡介苗接种的研究[J]. 中国防痨杂志, 2020, 42(8): 821-825.
[2]	杨鲁琦, 沈鸣逸, 沙巍, 陈颖盈, 王颖. 卡介苗的免疫保护机制及其改造策略进展[J]. 中国防痨杂志, 2020, 42(8): 863-868.
[3]	陈柠, 孙琳, 申阿东, 何秋水. 卡介苗接种后发生感染的可能原因研究现况[J]. 中国防痨杂志, 2020, 42(8): 869-873.
[4]	陈柠, 孙琳, 申阿东, 何秋水. 卡介苗非特异性保护作用研究进展[J]. 中国防痨杂志, 2020, 42(10): 1128-1133.
[5]	赵爱华,王国治,付丽丽,徐苗. 国内外结核病预防用卡介苗质量标准比较[J]. 中国防痨杂志, 2018, 40(10): 1125-1128.
[6]	董斯佳,袁立,陈诚,蒋伟利,赵琦. 细胞因子基因多态性与肺结核并发糖尿病易感性的相关性研究[J]. 中国防痨杂志, 2018, 40(10): 1051-1059.
[7]	王丁丁，付丽丽，寇丽杰，等.. 豚鼠模型卡介苗接种所引发的淋巴结炎致病菌的分离和鉴定[J]. 中国防痨杂志, 2016, 38(3): 209-214.
[8]	赵爱华，徐苗，郑素华，等. 针对世界卫生组织对卡介苗两次文件立场观点差异的探讨[J]. 中国防痨杂志, 2016, 38(2): 87-89.
[9]	赖赛麟，孙毅凡，陈涛，杨晓，周琳，钟球. γ干扰素活化的THP-1源巨噬细胞杀伤荧光BCG的实验研究[J]. 中国防痨杂志, 2014, 36(5): 362-368.
[10]	康万里，凌颖，徐苗，赵爱华，都伟欣，陈鸣，高铁杰，张治国，胡代玉，冯先惠，郭菁，张海霞，何菊，刘冠，李海英，吴新悦，王翠芝，郑素华，王国治. 皮内注射卡介苗接种效果的评价[J]. 中国防痨杂志, 2014, 36(4): 248-252.
[11]	张占军，姚岚，唐神结. 慢性阻塞性肺疾病合并肺结核患者部分细胞因子水平的表达及其意义[J]. 中国防痨杂志, 2014, 36(3): 189-193.
[12]	卢锦标，赵爱华，王国治，徐苗. 对我国结核病免疫预防策略的探讨[J]. 中国防痨杂志, 2014, 36(11): 927-929.
[13]	赵爱华，寇丽杰，徐苗，王国治. 我国皮内注射用卡介苗使用说明书修订意见[J]. 中国防痨杂志, 2014, 36(10): 863-867.
[14]	柴雪敏黎友伦. 结核分枝杆菌Ag85复合物相关疫苗研究进展[J]. 中国防痨杂志, 2013, 35(4): 282-285.
[15]	邢爱英刘忠泉刘洋贾红彦李自慧郑晓静曹廷明杜凤娇杜博平古淑香张宗德. 结核分枝杆菌Rpf-DNA疫苗对Mtb感染小鼠免疫保护作用的研究[J]. 中国防痨杂志, 2012, 34(8): 532-537.