甲型乙型肝炎联合疫苗诱导小鼠免疫应答特点的研究

A Study on the Immune Response Induced by Hepatitis A and B Combined Vaccine in Mice

目的比较甲型乙型肝炎联合疫苗(Hepatitis A and B Combined Vaccine,HepAB)与重组乙型肝炎(乙肝)疫苗(酿酒酵母)(Hepatitis B Vaccine Made by Recombinant Deoxyribonucleic Acid Techniques in SaccharomycesCerevisiae Yeast,HepB-SCY)、甲型肝炎(甲肝)灭活疫苗(Hepatitis A Virus Inactivated Vaccine,HepA-Ⅰ)诱导小鼠细胞免疫和体液免疫应答的特点。方法 4组无特定病原体的BALB/c小鼠(H-2d,6～8周龄,16～18克,雌性),分别皮下接种HepAB[含甲肝抗原150单位(Unit,U)、乙肝抗原3微克(μg)]、HepB-SCY(含乙肝抗原3μg)、HepA-Ⅰ(含甲肝抗原150U)及铝佐剂对照,于免疫后14d分离脾单个核细胞(Mononuclear Cell,MNC),应用酶联免疫斑点试验(Enzyme-linked Immunospot Assay,ELISPOT)测定MNC体外刺激后所产生的细胞因子干扰素(Interferon,IFN)-γ、白细胞介素(Interleukin,IL)-2斑点形成细胞数(Spots-forming Cell,SFC);同时应用酶联免疫吸附试验(Enzyme-linked Immunosorbent Assay,ELISA)检测免疫后脾MNC培养上清中诱导分泌抗原特异性IFN-γ细胞因子的含量;分别于免疫后14d、28d检测小鼠血清抗甲肝病毒抗体(Antibody to Hepatitis AVirus,Anti-HAV)IgG和抗乙肝病毒表面抗原抗体(Antibody to Hepatitis B Virus Surface Antigen,Anti-HBs)水平。结果应用ELISPOT法检测,HepAB免疫小鼠后诱导乙肝抗原特异性IL-2分泌水平(50%)高于HepB-SCY(20%),HepAB组诱导甲肝抗原特异性IFN-γ阳转率(70%)高于HepA-Ⅰ组(30%),且HepAB诱导乙肝抗原特异性IFN-γ分泌水平(80%)均显著高于HepB-SCY组(30%)(P=0.03489)。应用ELISA法检测HepAB免疫后脾MNC培养诱导分泌甲肝抗原特异性IFN-γ阳转率(80%),也显著高于HepA-Ⅰ组(20%)(P=0.01151)。HepAB组于免疫14d、28d诱导的Anti-HBs抗体滴度分别为93.6毫国际单位/毫升(mIU/ml)和476.8 mIU/ml,均高于HepB-SCY组的26.5 mIU/ml和237.8 mIU/ml。结论两种细胞免疫评价方法结果均表明,HepAB诱导抗原特异性细胞免疫应答反应优于HepB-SCY、HepA-Ⅰ;HepAB与HepB-SCY相比较,在诱导Anti-HBs水平方面也同样具有优势。

Objective To compare the cellular and humoral immune responses to the hepatitis A and B combined vaccine（HepAB）with hepatitis B vaccine made by recombinant deoxyribonucleic acid techniques in saccharomyces cerevisiae yeast（HepB-SCY）and hepatitis A virus（HAV）inactivated vaccine（HepA-I）in immunized mice.Methods Levels of interferon（IFN）-γ and interleukin（IL）-2 secreted by splenic mononuclear cells（MNC）of the vaccinated mice were detected by enzyme-linked immunospot method（ELISPOT）after in vitro stimulation with hepatitis B virus surface antigen（HBsAg）major histocompatibility complex（MHC）class I peptide S28-39,HBsAg or purified HAV,respectively.The cytokine concentration of IFN-γ secreted by MNCs of mice was detected by enzyme-linked immunosorbent assay（ELISA）method after stimulation with HBsAg or purified HAV.Levels of antibody to HBsAg（anti-HBs）and antibody to HAV（anti-HAV）were measured by the Abbott ARCHITECT Diagnostic kit.Results The HBsAg specific SFCs of IFN-γ and IL-2 secreted by MNCs of HepAB were higher than those of the HepB-SCY after in vitro stimulation with HBsAg MHC class I peptide S28-39 or HBsAg.The SFCs of IFN-γ,secreted by MNCs of HepAB were higher than those of HepA-I after in vitro stimulation with purified HAV.The cytokine concentration of IFN-γ,secreted by MNCs of HepAB was also higher than that of monovalent vaccine after stimulation with HBsAg or purified HAV.At different time,level of anti-HBs of HepAB were higher than that of HepB-SCY（93.6mIU/ml,476.8mIU/ml;26.5mIU/ml,237.8 mIU/ml）.Conclusion Results of two kinds of cellular immune assays showed that responses to HepAB were higher than those to monovalent vaccines.The combined vaccine also induces higher level of anti-HBs.