重组乙型肝炎病毒核心抗原的免疫原性及其对乙型肝炎病毒表面抗原的免疫增强作用

目的探讨重组乙型肝炎病毒核心抗原(HBcAg)的免疫原性及其对重组乙型肝炎病毒表面抗原(HBsAg)的免疫增强作用。方法用透射电镜观察HBcAg、HBsAg及HBcAg+HBsAg混合抗原病毒样颗粒(Virus-like particle,VLP)的形态。将小鼠随机分为6组:阴性对照组(NS)、HBcAg组(C)、HBsAg组(S)、HBsAg+铝佐剂组(S+Al)、HBsAg+HBcAg混合组(S+C)及HBsAg+HBcAg+铝佐剂组(S+C+Al),用相应抗原免疫各组小鼠,分别于第0天、第14天经小鼠双侧大腿肌肉进行初次和加强免疫1次,间接ELISA法检测各组小鼠血清中HBsAg和HBcAg的特异性抗体水平,分析HBsAg特异性抗体亚类,ELISPOT法检测各组小鼠脾细胞中HBsAg特异性IFNγ、HBcAg特异性IFNγ及HBsAg特异性IL-4水平。结果电镜观察可见HBsAg和HBcAg均呈大小均一的VLP结构,二者混合后有聚集现象。初次免疫后,S+C组的HBsAg特异性抗体水平与S组差异无统计学意义(P=0.074),加强免疫后,S+C组HBsAg特异性抗体水平明显高于S组(P=0.002),C组和S+C组均可产生高水平HBcAg特异性抗体。S组HBsAg抗体亚类以IgG1为主,S+C组以IgG2a为主。S+C组HBsAg特异性IFNγ斑点数明显高于S组、S+Al组和S+C+Al组(P<0.05),C组与S+C组HBcAg特异性IFNγ斑点数均较高,与S+C+Al组比较,S+Al组HBsAg特异性IL-4斑点数明显升高(P=0.026)。结论 HBcAg具有良好的免疫原性,可增强HBsAg的免疫反应,使反应向Th1方向极化,为治疗性乙肝疫苗的研发奠定了基础。

Immunogenicity of recombinant hepatitis B core antigen and its immunopotentiation to hepatitis B surface antigen

Objective To evaluate the immunogenicity of hepatitis B core antigen(HBcAg)as well as its immunopotentiation to hepatitis B surface antigen(HBsAg).Methods The morphologies of HBcAg,HBsAg and virus-like particles(VLPs)of HBcAg+HBsAg were observed by electron microscopy.Mice were randomly divided into negative control(normal saline,NS),HBcAg(C),HBsAg(S),HBsAg+aluminum adjuvant(S+A)l,HBsAg+HBcAg(S+C)and HBsAg+HBcAg+aluminum adjuvant(S+C+A)l groups,and immunized i.m.with the corresponding antigens on days 0 and 14 respectively,of which the sera were collected and determined for anit-HBs,anti-HBc and antibody subclass against HBsAg.Meanwhile,the splenocytes of mice were collected and determined for IFNγ levels against HBsAg and HBcAg as well as IL-4 level against HBsAg.Results Elec-tron microscopy showed that both HBsAg and HBcAg formed VLPs with uniform structures,while the VLPs of mixed antigen were aggregated.The anti-HBs levels after the first immunization showed no significant difference in S+C and S groups(P = 0.074),while those after the second immunization were significantly higher in S+C group than in S group(P = 0.002).High anti-HBc levels were induced in both C and S+C groups.The antibody subclasses against HBsAg in S group were mainly IgG1,while those in S+C group were mainly IgG2a.The number of spots formed by IFNγ against HBsAg in S+C group was significantly larger than those in S,S+Al and S+C+Al groups(P < 0.05).Both the numbers of spots formed by IFNγ against HBcAg were large in C and S+C groups.However,the numbers of spots formed by IL-4 against HBsAg were higher in S+C+Al group than in S+Al group(P = 0.026).Conclusion HBcAg showed good immunogenicity,which enhanced the immune response of HBsAg and promoted the Th1 polarization of the response.It laid a foundation of development of therapeutic HB vaccine.