重组乙型肝炎疫苗和乙型肝炎表面抗原诱导细胞免疫应答  

Cellular immune responses of recombinant hepatitis B （rHB） vaccine and HBsAg derived from Hansenular polymorpha cells

作　　者：胡忠玉[1] 何鹏[1] 张瑞[1] 方鑫[1] 朱凤才[2] 邱少辉[1] 李河民[1] 汪华[2] 梁争论[1] 庄辉[3] 

机构地区：[1]中国药品生物制品检定所,北京100050 [2]江苏省疾病预防控制中心 [3]北京大学医学部

出　　处：《中华流行病学杂志》2008年第7期706-711,共6页Chinese Journal of Epidemiology

基　　金：国家“十五”科技攻关课题资助项目（2004BA718802）

摘　　要：目的 探讨重组乙型肝炎（乙肝）疫苗和汉逊酵母菌表达的乙肝表面抗原（rHBsAg）诱导动物和人体细胞免疫应答的动态变化。方法 应用不同剂量rHBsAg免疫小鼠（BALB／c，H-2^d），酶联免疫斑点法（ELISPOT）检测小鼠脾单个核细胞（MNC）体外刺激诱生IFN-γ的水平；单剂rHBsAg免疫后不同时间检测MNC、CD8^＋T淋巴细胞产生IFN-γ水平；同时测定免疫后不同时间细胞毒T淋巴细胞反应（CTL）活性；检测免疫单剂和多剂乙肝疫苗后小鼠MNC分泌IFN-γ、IL-2、IL-5和抗-HBs抗体水平；对乙肝感染标志阴性的4名成年人按0、1、2个月程序接种乙肝疫苗，检测免疫后外周血单个核细胞（PBMC）、CD；T淋巴细胞分泌IFN-γ、IL-2和IL-4水平以及抗体动态。结果 应用ELISPOT法，汉逊rHBsAg免疫小鼠7d时，可检测到IFN-γ应答，14d时达到高峰；CTL在免疫后7d时可检出，峰值位于28d。在1～8ug免疫剂量范围内，MNCs IFN-γ免疫应答与剂量呈显著正相关（阳转率：r=0．951，P=0．049〈0．05；SFC：r=0．996，P=0．000〈0．05），在1～4ug免疫剂量范围内，CD8^＋T淋巴细胞的IFN-γ与剂量也呈显著正相关（r=0．999，P=0．025〈0．05）；三剂乙肝疫苗免疫小鼠诱生细胞免疫和体液免疫应答水平均显著高于单剂（P值均〈0．05）；成年人接种汉逊乙肝疫苗后不同个体间细胞免疫和体液免疫应答的模式不同，IL-2、IL-4应答与个体抗体滴度有关。结论 用ELISPOT法成功测定了汉逊HBsAg免疫小鼠后不同淋巴细胞的细胞免疫剂量效应和CTL时间动态变化，并分析了成年人的细胞免疫应答特点，为规范疫苗细胞免疫评价提供了基础。Objective To study the kinetics of immune response in mice and human immunized with rHB vaccine or rHBsAg derived from yeast cells （Hansenula polymorpha）. Methods With different doses, the level of IFN-γ secreted by spleen mononuclear cells （MNC） including CD8^＋ T cells by MACs of mice were detected by enzyme-linked immunospot （ELISPOT） methods after stimulation in vitro with HBsAg MHC class I peptide S28-39, respectively. At serial time points, the immunized mice were detected for IFN-γ by ELISPOT as above and for the lymphocytotoxicity test （CTL） by specific lysis assay. The levels of IFN-γ, IL-2, IL-5 and anti-HBs in mice induced by rHB vaccine were detected after single or three doses. Four adults were vaccinated with rHB vaccine according to 0, 1 and 2 month schedule. The peripheral blood mononuclear cells （PBMCs） were collected at the 3, 8, 21, 34 and 65 days after the first dose. The CD8^＋ T cells with high purity obtained by sorting from PBMCs were stimulated with rHBsAg or HBsAg peptides. The SFC of IFN-γ, IL-2 and IL-4 of CD4^＋ and CD8^＋ T cells were determined by ELISPOT. Results The cytokine of IFN-γ became detectable on day 7 and its peak value appeared on day 14 by ELISPOT. The CTL was detected on day 7 and the maximum lysis of CTL appeared on day 28. The cellular immune response of IFN-γ of MNCs were significantly correlated with the doses vaccinated from 1 ug to 8 ug （rpositive rates =0.951 ,Ppositive rates = 0.049〈0.05; rSFC = 0.996, PSFC = 0.000〈0.05）. IFN-γ SFC of CDs T cells were significantly associated with the doses from 1 ug to 4 ug （ r = 0. 999, P = 0. 025 〈 0.05）. The HBsAg specific cellular immune and humoral responses of mice immunized with three doses were significantly higher than that with a single dose （P〈 0.05）. The characteristics of IFN-γ, IL-2 and IL-4 of CD4^＋ and CD8^＋ T cells were variable between individuals immunized with the same rHB vaccine. The level of IL-2 and IL-4 of responders were significantly related to

关 键 词：乙型肝炎疫苗 免疫 细胞 乙型肝炎表面抗原 

分 类 号：R392[医药卫生—免疫学]