佐剂论文：不同疫苗佐剂对OVA诱生CTL免疫应答影响汇总

佐剂论文：不同疫苗佐剂对OVA诱生CTL免疫应答的影响

【中文摘要】CD8~+ CTL反应在肿瘤和慢性感染等疾病的治疗中具有重要作用,然而在研究中多使用DNA疫苗和载体活疫苗等通过内源性抗原加工提呈途径诱生细胞免疫,往往具有安全性差、免疫效果不理想和抗载体效应等缺点。蛋白和多肽等外源性抗原能够通过交叉提呈和交叉致敏诱生CD8~+ CTL反应,但需要有效的疫苗佐剂来增强抗原提呈和共刺激信号并提供Th1类细胞因子环境。本论文初步评价了新型疫苗佐剂CpG-ODN与Al(OH)3或Montanide ISA 720等组成的复合佐剂在小鼠体内促进蛋白抗原通过交叉提呈和交叉致敏诱生CD8~+ CTL反应的能力,从而为设计基于蛋白和多肽抗原的新型治疗性疫苗提供理论基础。在本研究中,首先以鸡卵清蛋白(OVA)为模型抗原,分别以CpG-ODN、Al(OH)3、Montanide ISA 720、CpG-ODN + Al(OH)3和CpG-ODN + Montanide ISA 720为疫苗佐剂,肌肉注射免疫C57BL/6小鼠,以胞内细胞因子染色和体内CTL杀伤等方法评价细胞免疫效果。在此基础上,进一步通过ELISPOT、胞内细胞因子染色、体内CTL杀伤和李斯特菌攻击等方法评价了A、B和C三种具有不同化学结构和生物学活性的CpG-ODN与Al(OH)3组成的复合佐剂对OVA抗原的细胞免疫佐剂效应。两针免疫后结果表明,与无佐剂对照组相

比,Al(OH)3本身并不能有效诱生细胞免疫,CpG-ODN或Montanide ISA 720单独使用能够在一定程度上增强抗原特异性CD8~+ T细胞的IFN-γ分泌和CTL活性,但不增强抗原特异性CD4~+ T细胞反应。两种复

合佐剂具有比单佐剂更强的细胞免疫佐剂效应,其中CpG-ODN + Montanide ISA 720只能增强抗原特异性CD4~+和CD8~+ T细胞的IFN-γ分泌,而CpG-ODN + Al(OH)3不但能够增强抗原特异性CD4~+和CD8~+ T细胞的IFN-γ反应,还能够增强CD8~+ CTL反应。三种不同类型CpG-ODN的比较研究结果表明,B和C型具有相近的细胞免疫佐剂效应,不但能够促进抗原特异性CD4~+和CD8~+ T细胞的IFN-γ分泌,而且能够诱生抗原特异性CD8~+ CTL反应。与此不同,尽管A型CpG-ODN能够在一定程度上促进抗原特异性CD8~+ CTL反应,但并不增强抗原特异性CD4~+和CD8~+ T细胞的IFN-γ分泌。与此相一致,B和C型CpG-ODN在小鼠李斯特菌攻击试验中也显示了比A型CpG-ODN更强的抗原特异性免疫保护作用。综上所述,在三种类型CpG-ODN中,B或C型CpG-ODN与Al(OH)3组成的复合佐剂在小鼠体内具有更强的促进蛋白抗原通过交叉提呈和交叉致敏诱生CD8~+ CTL反应的能力。

【英文摘要】CD8~+ CTL response plays a critical role in immunotherapies against tumors and chronic infections. DNA vaccines and live vector vaccines were conventionally used to induce cellular immunity through the classical endogenous MHCⅠclass antigen-presenting pathway. Exogenous antigens, like proteins and peptides, can induce CD8~+ CTL response by means of cross-presentation and cross-priming. However, effective vaccine adjuvants are needed to enhance antigen presentation

and co-stimulate signals as well as provide the Th1 type cytokine milleu. In the present study, CpG-ODN alone, or in combination with Al(OH)3 or Montanide ISA 720, were evaluated for their capacity to promoting CD8~+ CTL response through cross-presentation and cross-priming of protein antigens. This study would provide implications for the rational design of novel therapeutic vaccines based on protein and peptide antigens.Firstly, C57BL/6 mice were immunized intramuscularly with chicken ovalbumin (OVA) as a model antigen, and CpG-ODN, Al(OH)3, Montanide ISA 720, CpG-ODN + Al (OH)3 or CpG-ODN + Montanide ISA 720 as adjuvants, respectively. The cellular immune responses were evaluated by intracellular cytokine staining assay and in vivo CTL killing assay. On this basis, in combination with Al(OH )3 as composite adjuvants, three CpG-ODN classes (A-, B- and C-class) with different chemical compositions and biological activities were further compared for their immuno-potentiating effects on cellular immune responses against OVA antigen.The results showed that after two immunizations, compared with the control group without adjuvant, Al(OH)3 alone was not able to effectively induce cellular immunity. In contrast, CpG-ODN or Montanide ISA 720 alone enhanced antigen-specific CD8~+ T cell response (as

indicated by IFN-γsecretion and CTL activity) to some extent. However, they were not able to enhance antigen-specific Th1 type CD4~+ T cell response. Both composite adjuvants showed more stronger adjuvant effects, in which CpG-ODN + Montanide ISA 720 could only enhance IFN-γ-secreting CD4~+ and CD8~+ T cell responses, while CpG-ODN + Al (OH) 3 could not only enhance IFN-γsecretion of CD4~+ and CD8~+ T, but also promote the CD8 + CTL responses.Of three different classes of CpG-ODN, the B- and C-class sequences exhibited similar adjuvant effects on cellular immune responses, not only promoting IFN-γsecretion by antigen-specific CD4~+ and CD8~+ T cells, but also inducing antigen-specific CD8~+ CTL response. In contrast, although the A-class CpG-ODN could weakly enhance antigen-specific CD8~+ T cell response regarding cytotoxic activity, they were not able to elicit antigen-specific, IFN-γ-secreting CD4~+ and CD8~+ T cells. Consistent with this, B- and C-class CpG-ODN also provided better antigen-specific immune protection against Listeria monocytogenes challenge.In conclusion, either B- or C-class CpG-ODN, in combination with Al(OH)3 as composite adjuvants, showed the strongest ability to promote CD8~+ CTL response in mice via cross-presentation and cross-priming of protein antigens.

【关键词】佐剂 CpG-ODN Th1型免疫应答 细胞毒性T淋巴细胞 细胞免疫

【英文关键词】adjuvant CpG-ODN Th1immune response CTL cellular immunity

【目录】不同疫苗佐剂对OVA诱生CTL免疫应答的影响摘要5-7

ABSTRACT7-8

文献综述11-20

第一章 交叉提呈和

1.1 CD8~+ CTL

1.3

CpG-ODN 在治疗性疫苗中的应用研究进展11-20在免疫治疗中的作用11-12

1.2 CD8~+ CTL 的诱生机制12

MHCⅠ类分子抗原提呈途径12-14类分子提呈途径12-13交叉提呈途径13-1414-15

1.3.1 经典的内源性抗原MHCⅠ

1.3.2 非经典的外源性抗原MHCⅠ类分子1.4 通过疫苗免疫诱生CD8~+ CTL 反应

1.5 CpG-ODN 促进蛋白抗原对CD8~+ CTL 的交叉提呈和交

1.5.1 CpG-ODN16-17

1.5.2 CpG-ODN 促进交叉提

叉致敏15-18

呈和交叉致敏的作用机制17-1818-1920-46

1.6 本课题研究内容

试验研究

1.7 治疗性疫苗的评价与展望19-20

第二章 CpG-ODN与不同佐剂组合促进OVA诱生 CTL反应的

2.1 材料20-22

2.1.1 仪器和设备20

2.1.2 主

研究20-32要试剂20-2122疫22

2.1.3 常用溶液配方21-222.1.4 实验动物

2.2.2 动物免

2.2 方法22-242.2.1 免疫原的制备22

2.2.3 小鼠脾细胞的制备222.2.4 抗原特异性T 细胞

2.2.6 体内CTL 杀

2.3.1

标记22-23伤试验23-24

2.2.5 CFSE 标记小鼠脾细胞232.2.7 统计学处理24

2.3 结果24-29