皮内注射用布氏菌活疫苗的免疫学评价  

Immunological evaluation on live Brucella vaccine for intradermal injection

作　　者：陈成[1,2] 魏东[1] 李恪梅[1] 付丽丽[1,2] 黄长江[2] 王国治[1,2] 

机构地区：[1]中国食品药品检定研究院结核病疫苗室,北京100050 [2]温州医科大学环境与公共卫生学院,浙江温州325035

出　　处：《中国生物制品学杂志》2015年第3期228-232,共5页Chinese Journal of Biologicals

基　　金："重大新药创制"科技重大专项(2014ZX09304311-002)

摘　　要：目的探讨布氏菌活疫苗皮内注射小鼠产生的体液免疫、细胞免疫应答及免疫保护力。方法将30只小鼠随机分为3组,每组10只,分别为低剂量组(5×107个/只)、高剂量组(2×108个/只)和生理盐水对照组,3组均经后肢皮内注射布氏菌活疫苗,0.1 ml/只。免疫4周后,摘眼球取血,分离血清,制备脾脏淋巴细胞,ELISA法检测小鼠血清中抗Br-PPD Ig G抗体效价;ELISPOT法检测分泌IFNγ、IL-4的脾脏淋巴细胞数;ELISA法检测体外再刺激后小鼠脾脏淋巴细胞分泌的IFNγ水平;流式细胞术对T细胞亚群进行分类。用羊布氏菌M5弱毒株攻击免疫小鼠,通过脾脏荷菌量评价布氏菌活疫苗的免疫保护作用。结果低、高剂量组小鼠血清中抗Br-PPD Ig G抗体效价均较高,几何平均滴度分别为642和557;低、高剂量组小鼠在Br-PPD抗原刺激下,分泌IFNγ、IL-4的脾脏淋巴细胞数以及脾脏淋巴细胞分泌的IFNγ含量,与对照组相比差异均有统计学意义(P均<0.05);CD4+IFNγ+、CD4+IL-4+比例均明显高于对照组,差异均有统计学意义(P均<0.05);低、高剂量组小鼠脾脏荷菌量为0,对照组小鼠为(5.13±0.16)log10 CFU。结论采用皮内注射布氏菌活疫苗的方式免疫小鼠后,能获得较强的体液免疫、细胞免疫应答及免疫保护力。Objective To evaluate the humoral and cellular immune responses as well as protective immunity of live Brucella vaccine for intradermal injection in mice. Methods Thirty mice were randomly divided into three groups, ten for each, and immunized i. d. with live Brucella vaccine at low（5 × 107 bacteria）and high（2 × 108 bacteria）and physiological saline, each in a volume of 0. 1 ml, respectively. Serum samples were collected 4 weeks after immunization, while the splenic lymphocytes were prepared. The Ig G titer against Br-PPD in sera was determined by ELISA, while the splenic lymphocytes secreting IFNγ and IL-4 were counted by ELISPOT. The IFNγ level secreted by mouse splenic lymphocytes after stimulation in vitro was determined by ELISA. The T cell subsets were tested by flow cytometry. The immunized mice were challenged with attenuated Brucella melitensis M5 strain, and the immune protection of vaccine was evaluated by the bacterial count in spleen. Results The GMTs of Ig G against Br-PPD in sera of mice immunized with vaccine at low and high dosages were 642 and 557 respectively. The counts of splenic lymphocytes secreting IFNγ and IL-4 as well as the secreted IFNγ contents after stimulation with Br-PPD of mice immunized with the vaccine at low and high dosages showed no significant difference with（each P〈0. 05）, while the proportions of CD4＋ IFN-γ＋ and CD4＋ IL-4＋ lymphocytes were significantly higher, than those in control group（each P〈0. 05）. Both the bacterial counts in spleens of mice immunized with vaccine at low and high dosages were 0, while that in control group was（5. 13 ± 0. 16） log10 CFU. Conclusion Live Brucella vaccine for intradermal injection showed strong humoral and cellular responses and protective immunity in mice.

关 键 词：布氏菌活疫苗 体液免疫 细胞免疫 

分 类 号：R378.5[医药卫生—病原生物学] R392.33[医药卫生—基础医学]