【作者】 张炜；

【导师】 瞿涤；

【作者基本信息】 复旦大学 ， 病原生物学， 2003， 博士

【摘要】 基因佐剂促进不同乙肝疫苗诱导免疫应答的动态学研究全球约有4亿的乙型肝炎病毒（HBV）慢性感染者，其中许多人发展为慢性肝脏疾病如肝硬化、肝癌等。由于目前尚无针对HBV持续性感染的有效药物，因此治疗性疫苗成为抗HBV感染领域中的一个研究热点。传统的蛋白疫苗诱导以IgG抗体为主的体液免疫应答，对慢性乙肝病毒感染作用有限。DNA 疫苗能诱生较强的特异性CD8+T细胞应答，但在大型哺乳动物和人类中所诱导的免疫应答强度相对较低。基因佐剂与疫苗共免疫是增强机体对疫苗的免疫应答或改变免疫应答类型的策略之一。本文拟研究不同基因佐剂对乙肝蛋白疫苗和DNA疫苗诱导的免疫应答的促进作用进行初步探讨。 已知典型的T细胞免疫应答分为三个期：⑴效应期 ⑵凋亡期 ⑶记忆期。基因佐剂通常着眼于疫苗诱导的T细胞免疫应答的三个时期，或是增强效应期T细胞扩增，或是减少效应期T细胞的凋亡，或是促进记忆性T细胞的形成，以增强疫苗的免疫效果。在T细胞免疫应答的效应期，T细胞的数量和质量首先取决于抗原递呈细胞（APC）呈递给T细胞的信号。由于树突状细胞（DC）是体内功能最强和最重要的抗原递呈细胞，能够显著活化初始T细胞进行增殖，因此增强DC的功能是增强治疗性疫苗效果的策略之一。细胞因子Flt3配体（FL）是影响DC的功能和数量的重要因素之一，可促使淋巴系来源的DC和髓系来源的DC大量扩增，因此FL作为基因佐剂时，可能对疫苗诱导的免疫应答的效应期产生影响。在T细胞免疫应答的记忆期，IL-15是记忆性CD8+T细胞产生和维持的关键因子。它是唯一能在体外直接诱导记忆性CD44hiCD8+T细胞增殖的细胞因子。因此IL-15作为基因佐剂，可能对疫苗诱导的免疫应答的记忆期产生影响。本论文以FL作为影响疫苗诱导的免疫应答效应期基因佐剂的代表，以IL-15作为影响影响疫苗诱导的免疫应答记忆期基因佐剂的代表，重点研究了基因佐剂对乙肝蛋白疫苗和乙肝DNA疫苗诱导的免疫应答的动态学影响。 第一章 乙肝病毒核心抗原 DNA疫苗诱导小鼠抗原特异性CD8+T细胞应答的动态研究<WP=6>为研究HBcDNA疫苗诱导的CD8+T细胞应答的动态变化，将HBV核心抗原（HBcAg）N端144个氨基酸的重组真核表达质粒DNA（简称pHBc144）免疫小鼠，利用细胞内细胞因子染色技术在DNA疫苗免疫后不同时间点定量检测小鼠的脾脏和PBMC中抗原特异性CD8+T细胞（CD8+IFN-γ+）占CD8+T细胞的百分比。结果显示：初次免疫后第8天，小鼠脾脏内0.10%±0.04%的CD8+T细胞是抗原特异性 CD8+T细胞，随后抗原特异性CD8+T细胞占CD8+T细胞的百分比逐渐上升，初次免疫后第14天，DNA疫苗诱导的特异性细胞免疫应答达到高峰，抗原特异性CD8+T细胞占CD8+T细胞总量的1.84%±0.56%，此后抗原特异性CD8+T细胞所占百分比逐渐减少，到初次免疫一年后抗原特异性CD8+T细胞仍占CD8+T细胞的0.28%±0.05%。加强免疫后第10天达到高峰，约占CD8+T细胞总数的3.60%±0.35%，约是初次免疫高峰的2倍，此后抗原特异性 CD8+T细胞的百分比再次回落，但下降的速度减缓，加强免疫后第30天抗原特异性CD8+T细胞仍占脾脏CD8+T细胞的1.17%±0.78%，明显高于初次免疫第30天抗原特异性CD8+T细胞的百分比，直至加强免疫后第60天抗原特异性CD8+T细胞的百分比才回落至0.63%。PBMC中IFN-γ+CD8+T细胞动态变化的趋势与脾脏特异性CD8+T细胞变化趋势相似，在初次免疫后第8天，小鼠外周血HBcAg特异性 CD8+T细胞占总CD8+T细胞的百分比低于检测水平（＜0.1%），初次免疫后第14天为特异性细胞免疫应答的高峰期，抗原特异性 CD8+T细胞占外周血总CD8+T细胞的0.23 %±0.21 %，此后抗原特异性CD8+T细胞占总CD8+T细胞的百分比逐渐减少，到初次免疫一年后小鼠外周血HBcAg特异性 CD8+T细胞占总CD8+T细胞的百分比低于检测水平（＜0.1%），加强免疫后第10天再次出现免疫应答高峰，抗原特异性的CD8+T细胞占外周血总CD8+T细胞的1.66%±0.02%，加强免疫后第30天外周血中抗原特异性CD8+T细胞约占总CD8+T细胞的0.45 %±0.28 %，直至加强免疫后第60天外周血抗原特异性CD8+T细胞回落至0.38%。尽管PBMCs中CD8+T细胞动态变化的趋势与脾脏相似，但同一时间点外周血中抗原特异性CD8+T细胞占CD8+T细胞总量的百分比明显少于脾脏。 第二章 IL-15表达质粒的构建及其对不同疫苗诱导的免疫应答的动态学影响 为探讨IL-15作为基因佐剂的可能性，我们首先从ConA刺激的人外周血单个核细胞抽提RNA，通过RT-PCR的方法，获得并克隆了人IL-15的cDNA ，并在Eco.li 中进行了表达，获得了分子量为26KD的融合蛋白，这与从核苷酸序列推测的分子量基本相同。通过CTLL-2淋巴细胞增殖试验证明了构建的IL-15真核表达质粒（简称pIL-15）具有生物学活性。<WP=7>为研究IL-15作为基因佐剂对乙肝蛋白疫苗诱导的免疫应答的动态学影响，我们将pIL-15与HBsAg共同免疫小鼠，结果显示：HBsAg + pIL-15组诱生的抗HBs效价高于HBsAg + pcDNA3免疫组（P ＜0.05）； 但HBsAg + pIL-15组IgG2a/ IgG1比值与HBsAg + pcDNA3组相比差别无显著性（P ＞0.05）。表明IL-15作为基因佐剂可增强乙肝蛋白疫苗诱导的体液免疫应更多还原

【Abstract】 The effect of genetic adjuvants on the kinetics of immune response to different HBV vaccinesIntroductionAn estimated 400 million people worldwide were chronically infected with HBV, and many of them developed into chronic diseases such as liver cirrhosis and hepatocellular carcinoma. At present there is no efficient drug gainst HBV persistent infection, and then the therapeutic vaccine become a hot spot. The conventional protein-based vaccine induces the humoral immune response which consists mainly of IgG, but it has limited effect on the chronic HBV infection. Although DNA vaccine could induce antigen-specific cytotoxic T lymphocytes, it has relatively low efficacy in large animal species and humans. Construction of genetic adjuvant is an alternative approach in which genes are co-delivered in order to modulate immune responses to vaccine. In this study, we discussed the effect of the different genetic adjuvants on the immune responses to HBV protein-based vaccines and DNA vaccines. T cell responses consist of three distinct phases: ⑴activation and expansion phase, ⑵death phase and ⑶memory phase. Genetic adjuvants might give intervention at any of these three phases: for example, it could increase the expansion of effector T cell populations, alternatively, it might be possible to intervene during the contraction phase to reduce effector T cell death, thereby increasing the percentageof memory T cells that formed.In the expansion phase, the quantities and qualities of T cells are determined by the signals given by antigen presenting cell (APC). Because dentritic cell (DC) is the most potent and important APC and could activate the na?ve T cell, enhancing the function of DC is one of the strategies to improve the efficacy of vaccine. Cytokine flt3 ligand (FL) is one of important factors which could influence the function and quantity of DC. It could induce the proliferation of the lymphoid and myeloid DC, so when used as an adjuvant, it might influence the expansion phase of immune response induced by vaccines.In the memory phase, IL-15 play a key role in the generation and maintenance of CD8+ memory T cells. It is the only one cytokine known that could induce the<WP=12>memory CD44hiCD8+T cell proliferation ex vivo. So when used as an adjuvant, it might influence the memory phase of the immune response induced by vaccines.In this study, we choose the FL as a representative of the genetic adjuvant which could affect the expansion phase induced by the vaccine, the IL-15 as the representative of the genetic adjuvant which could affect the memory phase, to investigate the effect of the genetic adjuvant on the kinetics of immune responses to HBV protein-based vaccines and DNA vaccines.Part I The kinetic and distribution analyses of CD8+ T Cells responses induced by hepatitis B virus core gene DNA vaccine by intracellular cytokine stainingTo study the kinetics of immune responses induced by HBV core gene DNA vaccines,100 μg of a recombinant expression plasmid DNA, harboring DNA encoding the 144 amino acids of the N-terminus of HBV core gene (abbreviated pHBc144) was used to immunize C57BL/6 mice. The percentage of antigen-specific CD8+ T cell (CD8+IFN-γ+T cells ) was detected by intracellular cytokine staining（ICCs）at various time point. On day 8 postimmunization，the percentage of antigen-specific T cells in spleen is 0.10%±0.04%.Then there was a rapid amplification of antigen-specific T cells and peaking at 14 days postimmunization（about 1.84%±0.56% of all splenic CD8+ T cells）, then antigen-specific CD8+ T cell decreased gradually，but remained detectable directly ex vivo for one year postinjection（about 0.28% of all splenic CD8+ T cells）. And after boosting, the percentage of antigen-specific CD8+ T cell rebounded and reached the peak on 10 days later（about 3.60%±0.35% of all splenic CD8+ T cells）, the value is 2 fold higher than the peak of first immunization, then the percentage declined slowly until by day 60 after second immunization it returned back to 0.32%?更多还原