【作者】 栾翔凌；

【导师】 辛绍杰； 貌盼勇；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2008， 博士

【摘要】 由于DNA疫苗在诱导机体特异性细胞免疫方面具有独特优势,已作为针对慢性乙型肝炎具有潜力的免疫治疗手段被广泛研究。如何加强DNA疫苗的效果是推动DNA疫苗发展的关键。其中一个研究方向就是通过选择佐剂和调整免疫程序加强DNA疫苗的保护效率。异源性载体prime/boost免疫策略是利用不同性质的载体表达同一个保护抗原,一般是DNA疫苗初次免疫(prime),然后用病毒载体加强(boost)。DNA疫苗抗原纯度高,持续表达低剂量的抗原,可多次重复免疫以及诱导高效价记忆性细胞毒性T淋巴细胞(CTL)反应,是理想的用于初次免疫的疫苗;重组病毒能高效表达目的抗原,模拟病毒感染诱导机体免疫的天然过程,能诱发多种细胞因子,而且有利于高亲和性抗体和T细胞受体的选择,可以有效地产生Th1型和CTL反应。白细胞介素18(IL-18)最早被称为IFN-γ诱导因子γ,它不仅能促进T细胞和NK细胞产生IFN-γ、IL-2和GM-CSF等细胞因子,并能增强Th1细胞和NK细胞表达Fas配体,从而介导细胞毒作用。本课题在构建含HBV不同抗原基因的重组MVA假病毒颗粒的基础上,采取DNA prime/VLP boost的免疫程序,辅以细胞因子IL-18作为佐剂,评价其治疗HBV慢性感染的可能价值。首先将表达乙型肝炎病毒PreS2-S、C、PreC/C等抗原基因,以及分别突变2个和4个核苷酸的PreC/C基因分别克隆入与痘苗病毒Ankara株(MVA)基因组同源的穿梭载体pSC11中,构建并筛选重组质粒,将所得转染MVA病毒经过9次传代培养,筛选分别携带这些目的基因的重组MVA病毒,通过PCR扩增证实重组病毒分别正确携带目的基因,并且在传代过程中成功形成单克隆,间接免疫荧光鉴定重组病毒可以被所表达抗原的特异性抗体识别,证实重组病毒正确表达目的抗原并具有良好的抗原性。大量制备DNA疫苗、重组病毒以及鼠白细胞介素-18(mIL-18)质粒,分别采用DNA疫苗单独免疫、rMVA单独免疫、DNA prime/rMVA boost、DNA prime/rMVAboost+mIL-18的免疫程序免疫C57BL/6小鼠,采用ELISA、ELISPOT方法评价体液免疫和细胞免疫的效果。结果显示,表达PreS2-S、C两种抗原的各组疫苗在抗体产生的时间和滴度方面均没有统计学差异,用ELISPOT检测IFN-γ的产生可知,所有免疫组均可引起针对特异性抗原的细胞免疫,rMVA单独免疫组强于DNA疫苗单独免疫组,弱于DNA prime/rMVA boost和DNA prime/rMVAboost+mIL-18组,DNA prime/rMVA boost和DNA prime/rMVA boost+mIL-18之间没有显著差别。研究表明,携带HBV保护性抗原基因的重组MVA病毒可以诱导针对抗原的特异性细胞免疫和体液免疫,将其与表达相同抗原的DNA疫苗联合,采用DNAprime/rMVA boost的免疫方案,可以诱导比两者单独应用更强的细胞免疫反应,实验结果将为治疗型HBV疫苗的应用研究提供科学依据。更多还原

【Abstract】 DNA vaccine,with a predominant advantage in inducing specific cellular immunity in vivo,DNA vaccine has been widely determined as a potential immunotherapy method for treating CHB.How to enhance the efficiency should be particularly focused on DNA vaccine discovery.One possible way is to adopt suitable immunoadjuvants as well as to optimize immunization strategies.The immunization strategy of the heterologous vector(prime/boost)is achieved by the co-expression of the same protective antigen with different vectors,i.e.prime immunization by a DNA vaccine(prime)and boost immunization by a viral vector (boost).As an ideal prime immunization vaccine,the DNA vaccine has the advantages of high purity,continuous expression of antigens with low dose, repeatable immunization and inducement of memory CTL with high titer.Similarly, recombinant virus can not only highly express the target antigen,simulate the inducement process of virus infection on body immunity,and induce various cytokines,but also be beneficial to the interaction of high-affinity antibodies with T cell receptors,and the generation of Th1 reaction and CTL reaction.Interleukin-18(IL-18),also called interferon-gamma inducing factor(IGIF), can not only promote T cells and NK cells to generate cytokines,such as IFN-γ,IL-2 and GM-CSF,but also strengthen the expression on Fas ligand in Th1 cells and NK cells in order to mediate cytotoxicity and promote the development of Th1 cells.In this paper,the genes expressing PreS2-S、C、PreC/C and two mutanted PreC/C of HBV adr subtype were inserted into a shuttle vector pSC11,which is homologous with modified vaccinia virus Ankara(MVA),to construct and screen recombinant plasmid.The recombinant plasmid was then transfected into MVA virus. Recombinant MVA viruses respectively carrying the genes were screened after subculture of nine generations.Besides,monoclonal virus without wild virus was obtained during the subculture process.Indirect immunofluorescent analysis was used to identify the recombinant virus.The results showed that the recombinant virus could be recognized by the specific antibody against the antigen expressed by the recombinant virus.It was thus proved that the recombinant virus could express target antigen correctly and had good antigenicity.The DNA vaccine,the recombinant virus and mouse IL-18(mIL-18)were prepared on large scale.C57BL/6 mice were immunized by different immunization processes,including single DNA vaccine immunization,single rMVA immunization, DNA prime/rMVA boost and DNA prime/rMVA boost + mIL-18.The effects of humoral immunity and cellular immunity were evaluated by ELISA and ELISPOT. The results showed that there was no statistic difference among the vaccine groups capable of expressing two antigens,in the respect of antibody generation time and titer.The ELISPOT detection of IFN-γshowed that all immunization groups could induce cellular immunity against the specific antigen.The effect of the single rMVA immunization group was better than that of the single DNA vaccine immunization group,while weaker than that of the DNA prime/rMVA boost group and the DNA prime/rMVA boost + mIL-18 group.There was no significant difference between the DNA prime/rMVA boost group and the DNA prime/rMVA boost + mIL-18 group.The recombinant MVA virus induced specific cellular and humoral immune response toward HBV antigen.With the strategy of DNA prime/rMVA boost, stronger cellular immune were induced than DNA vaccine or rMVA alone,which provides basic evidence for the application of gene therapy for HBV.更多还原