复合疫苗佐剂促进蛋白抗原通过交叉提呈和交叉致敏诱生CD8^+CTL反应的研究

Studies on vaccine adjuvants to promote CD8^+ CTL response through cross-presentation and cross-priming of protein antigens

目的研究CpG寡聚脱氧核苷酸(CpG-oligodeoxynucleotides,CpG-ODN)与A(lOH)3或Montanide ISA720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。方法以鸡卵清蛋白(Ovalbumin,OVA)为抗原,分别以CpG X1、A(l OH)(3即Alum)、Montanide ISA720、CpG X1+Alum和CpG X1+Montanide ISA720为疫苗佐剂,分别于0和4周经肌肉注射免疫C57BL/6小鼠,体积均为100μl,分别含20μg OVA、20μg CpG X1、74μl Montanide ISA720和/或100μg Alum。通过胞内细胞因子染色和体内CTL杀伤试验评价不同佐剂对细胞免疫应答的影响,通过表达OVA的黑色素瘤和李斯特菌攻击模型评价不同佐剂在免疫预防和免疫治疗中的作用。结果与OVA组相比,A(lOH)3本身不能有效诱生小鼠的细胞免疫应答;CpG X1或Montanide ISA720单独使用能够在一定程度上增强抗原特异性CD8+T细胞的IFNγ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 X1+MontanideISA720只能增强抗原特异性CD4+和CD8+T细胞的IFNγ分泌,而CpG X1+Alum不仅能够增强抗原特异性CD4+和CD8+T细胞的IFNγ分泌,还能够增强CD8+CTL的杀伤活性。在黑色素瘤和李斯特菌攻击模型中,CpG X1+Alum佐剂显示出良好的预防和治疗效果。结论 CpG X1与A(lOH)3组成的复合佐剂能够有效促进蛋白抗原通过交叉提呈和交叉致敏诱生功能性CD8+CTL反应。

Objective CpG-ODN alone,or in combination with Al（OH）3 or Montanide ISA720,were evaluated for their capacity to promote 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.Methods C57BL / 6 mice were immunized intramuscularly with chicken ovalbumin（OVA） as a model antigen,alone or in combination with CpG-ODN,Al（OH）3,Montanide ISA720,CpG-ODN ＋ Al（OH）3 or CpG-ODN ＋ Montanide ISA720 as adjuvants,respectively.The protective cellular immune responses were evaluated by intracellular cytokine staining assay,in vivo CTL killing assay,and animal challenge with OVA-expressing melanoma and Listeria monocytogenes.Results 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 ISA720 alone enhanced antigen-specific CD8^＋ T cell response to some extent,as indicated by IFNγ secretion and CTL activity.However,they were not able to enhance antigen-specific Th1 type CD4^＋ T cell response.Although both composite adjuvants showed stronger adjuvant effects,CpG-ODN ＋ Montanide ISA720 could only enhance IFNγ-secreting CD4^＋ and CD8^＋ T cell responses,while CpG-ODN ＋ Al（OH）3 enhanced not only IFNγ secreting CD4^＋ and CD8^＋ T cell responses but also CD8^＋ CTL activity.Consistent with this,CpG-ODN ＋ Al（OH）3 as an adjuvant for protein antigen showed promising preventive and therapeutic effects in both melanoma and listeria challenge experiments.Conclusion CpG-ODN ＋ Al（OH）3 as a composite adjuvant can enhance antigen-specific CD8^＋ CTL response through cross-presentation and cross-priming of protein antigens.