基于T细胞表位的HBV DNA疫苗及结核杆菌热休克蛋白70的佐剂功能的研究

发布时间：2018-06-05 04:16

本文选题：基于 + 细胞　；参考：《中国科学技术大学》2006年博士论文

【摘要】：尽管预防性亚单位重组乙肝疫苗和一些治疗性药物在临床上已经广泛的应用，乙肝病毒(Hepatitis B Virus，HBV)感染目前仍然是严重一个威胁人类健康的尚未攻克的疾病。由于DNA疫苗具有诱导体液免疫和细胞免疫的独特的能力，因此为预防和治疗HBV感染提供了一个新的发展方向。近年来进行的Ⅰ期临床实验结果证明HBV DNA疫苗可以在健康人群中诱导保护性反应：在慢性HBV感染人群中可以诱导特异性的T细胞反应和清除HBVDNA。然而，这些临床实验中使用的HBV DNA疫苗只编码单一的抗原蛋白——S抗原或PreS2/S抗原。而且由于DNA疫苗独特的抗原表达和递呈途径，它的免疫原性仍然需要进一步的改进。因此，我们根据HBV表面抗原(HBsAg)上的T细胞表位，设计了一个包含多个细胞毒性T细胞(CTL)表位和辅助性T细胞(Th)表位的多表位DNA疫苗，并研究了它的功能，同时研究了结核杆菌热休克蛋白(Heat Shock Protein，HSP)HSP70的分子佐剂功能。结果表明，在H-2~d和H-2~b小鼠模型中，多表位HBV DNA疫苗诱导了一个比单一抗原基因的HBsAg DNA疫苗更强的HBsAg特异性CTL反应。然而，在H-2~d小鼠模型中，D~d-限制性表位的CTL反应明显弱于L~d-限制性表位的CTL反应。HSP70作为分子佐剂可以明显改善多表位DNA疫苗的有效性。当HSP70融合多表位抗原时，不仅HBsAg抗原特异性的CTL反应被增强了，而且各个表位特异性(L~d、K~d和K~b)的CTL反应也明显的增强了。在HBV转基因小鼠模型中，多表位DNA疫苗免疫可以明显的清除血液中的HBsAg，并显著下调了HBV DNA的水平。因此，，我们的研究表明基于T细胞表位的多表位DNA疫苗在治疗慢性HBV感染方面是一个可以考虑的方案。以往的研究表明，尽管HSP70可以改善DNA疫苗的有效性，但是由于HSP70是一个高度进化保守的分子，因此有引起自身免疫反应的可能性。在这个研究中，我们选择了两个截短的HSP70分子——N′-端的HSP70_(1-360)和C′-端HSP70_(359-610)，研究它们对于HBV DNA疫苗引起的抗原特异性的免疫反应的调节作用。实验结果表明，只有HSP70_(359-610)融合的HBV DNA疫苗诱导了一个增强的HBsAg特异性的抗体反应，同时，这个融合疫苗并没有诱导HSP70的抗体反应。有趣的是，HSP70_(359-610)不仅仅调节了HBsAg特异性的CTL反应，还在H-2~d小鼠模型中，明显增强了L~d-限制性表位的CTL反应和D~d-限制性表位的CTL反应。同时，HSP70_(359-610)融合DNA疫苗免疫调节Th反应平衡偏向于Th1方向。在转基因小鼠模型中，HSP70_(359-610)促进了HBV DNA免疫引起的HBsAg清除和HBV DNA下调。我们的研究结果表明，在HBV DNA疫苗设计中，截短的结核杆菌热休克蛋白HSP70分子——HSP70_(359-610)，可以替代完整的HSP70分子作为分子佐剂。
[Abstract]:Although prophylactic subunit recombinant hepatitis B vaccine and some therapeutic drugs have been widely used in clinic, hepatitis B virus (HBV) infection is still a serious threat to human health. Because of the unique ability of inducing humoral and cellular immunity, DNA vaccine provides a new direction for the prevention and treatment of HBV infection. The results of phase I clinical trials in recent years have shown that HBV DNA vaccine can induce protective response in healthy population: in chronic HBV infected population, it can induce specific T cell response and eliminate HBV DNA. However, the HBV DNA vaccine used in these clinical trials encodes only a single antigen protein, S antigen or PreS2/S antigen. Moreover, the immunogenicity of DNA vaccine needs further improvement because of its unique antigen expression and presentation. Therefore, based on the T cell epitopes on HBV surface antigen, we designed a multiepitope DNA vaccine containing multiple cytotoxic T cell HBV epitopes and helper T cell epitopes, and studied its function. At the same time, the function of molecular adjuvant of heat shock protein HSP70 from Mycobacterium tuberculosis was studied. The results showed that the multiepitope HBV DNA vaccine induced a stronger HBsAg specific CTL reaction than the HBsAg DNA vaccine with single antigen gene. However, the CTL response of Dnd- restricted epitope was significantly weaker than that of Lnd- restricted epitope CTL reaction. HSP70 as a molecular adjuvant could significantly improve the effectiveness of multiepitope DNA vaccine. When HSP70 fused polyepitope antigen, not only the specific CTL reaction of HBsAg antigen was enhanced, but also the CTL reaction of each epitope specific, LndndKnd and Knb) was obviously enhanced. In HBV transgenic mice, the multiple epitope DNA vaccine immunization could significantly eliminate HBsAg in blood, and significantly down-regulate the level of HBV DNA. Therefore, our study shows that multiepitope DNA vaccine based on T cell epitopes is a potential alternative for the treatment of chronic HBV infection. Previous studies have shown that although HSP70 can improve the effectiveness of DNA vaccines, HSP70 is a highly evolutionarily conserved molecule, so it is possible to induce an autoimmune response. In this study, we selected two truncated HSP70 molecules, HSP701-360) and CS-terminal HSP70- (359-610), to study their regulatory effects on the antigen-specific immune response induced by HBV DNA vaccine. The results showed that only HSP70- (359-610) HBV DNA vaccine induced an enhanced HBsAg specific antibody response, and the fusion vaccine did not induce HSP70 antibody response. It is interesting to note that HSP70359-610) not only regulates the specific CTL response of HBsAg, but also enhances the CTL response of Lnd- restricted epitope and the CTL reaction of Dnd- restricted epitope in H-2d mouse model. At the same time, the balance of Th response in immunomodulation of DNA vaccine was inclined to the direction of Th1. In transgenic mice model, HSP70 tippon 359-610) promoted HBsAg clearance and HBV DNA down-regulation induced by HBV DNA immunization. Our results show that the truncated HSP70 molecule of the heat shock protein (HSP70) of Mycobacterium tuberculosis can replace the complete HSP70 molecule as a molecular adjuvant in the design of HBV DNA vaccine.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

【共引文献】