减毒鼠伤寒沙门菌为载体的幽门螺杆菌治疗性疫苗实验研究

发布时间：2018-07-07 11:54

本文选题：幽门螺杆菌 + 表位疫苗　；参考：《第三军医大学》2011年博士论文

【摘要】：幽门螺杆菌(H.pylori)是一种定植于人胃和十二指肠粘膜的螺旋形革兰阴性菌,主要导致慢性胃炎、消化性溃疡和胃癌。目前临床治疗H.pylori感染的方案,多采用包括两种抗生素和一种质子泵抑制剂的“三联疗法”,在一定程度上能清除H.pylori感染并治愈溃疡。虽然抗生素在短期内具有较好的根除疗效,但抗生素的滥用已导致H.pylori耐药率逐年上升。H.pylori高耐药性是导致根除治疗失败的最主要原因,长此以往,可能出现H.pylori感染无药可治的严重后果。此外,抗生素疗法还存在治疗后易复发、可再感染和肠道菌群微生态失衡等不足,大大限制了其使用。在临床上H.pylori高耐药性的条件下,新抗生素因其研发周期长难以满足临床用药需求,因此,寻求非抗生素的替代疗法已迫在眉睫。目前,国际上针对H.pylori感染已广泛开展免疫治疗研究,动物和人体试验结果表明,免疫治疗具有清除H.pylori的效果,提示治疗性疫苗有希望成为治疗或辅助抗生素治疗H.pylori感染的方法。H.pylori全菌疫苗(包括灭活全菌和全菌裂解物为抗原的疫苗)非常有效。然而,这种以全菌组分作为抗原的疫苗方式有着严重的局限性,比如菌体中LPS和Lewis抗原可能导致内毒素血症、炎症反应和自身免疫性疾病等严重后果。以重组亚单位为抗原的疫苗和表位疫苗都表现出较好的抗H.pylori作用。目前已经确定了一些H.pylori保护性抗原(如UreB、CagA和VacA等),已作为候选抗原用于开发H.pylori疫苗。自然感染H.pylor后,引起Th1细胞活化,导致胃粘膜发生炎症损伤甚至溃疡。机体通过Treg细胞抑制胃粘膜过强的免疫应答,这有利于胃粘膜炎症损伤的修复,但同时使免疫系统对H.pylor处于免疫耐受状态。CD4+T细胞在抗H.pylori保护性免疫中发挥重要作用的观点已被广泛接受,大多数研究表明:高水平Th1偏向应答是保护性免疫和清除H.pylori感染必需的。治疗性疫苗要立足于打破机体对H.pylor的免疫耐受,诱导以Th1为主的CD4细胞免疫应答,产生高水平IFN-γ等细胞因子,进一步激活巨噬细胞等天然免疫,同时诱导粘膜sIgA应答,发挥清除H.pylori感染和预防再感染的作用。口服沙门菌载体疫苗可以诱导典型的Th1型应答,同时还能诱导显著的粘膜sIgA产生。此外,沙门菌还可以通过微皱细胞(microfold cell,M细胞)的转胞吞作用或通过树突状细胞(dendritic cell,DC)直接提呈抗原诱导粘膜免疫应答。已有大量文献报道,表达外源蛋白抗原的减毒沙门菌活载体疫苗用于抗不同的病原体(包括病毒、细菌和寄生虫)感染,在动物模型实验中均取得了良好的效果。相关的研究表明,口服免疫重组沙门菌疫苗具有安全性好,免疫原性良好的特点。全长的H.pylori蛋白分子作为疫苗抗原含有非必要的抗原表位,而且分子量较大,难以表达和纯化。而融合表位抗原易于表达和纯化,融合蛋白疫苗是一种新的疫苗策略,可用于增强对多抗原组分的优势抗原表位的特异性免疫应答。【研究目的】 1.本研究拟在H.pylori感染的小鼠模型体内评价表达不同抗原表位融合蛋白的减毒沙门菌载体疫苗的治疗效果。 2.初步明确Th1和Th2细胞应答在疫苗免疫清除H.pylori感染中的作用。【研究方法】 1.构建表达H.pylori按不同顺序排列的CagA、VacA和UreB抗原表位融合蛋白的减毒沙门菌载体疫苗,在H.pylori感染的小鼠模型体内评价其疗效。 2.Real-time PCR检测免疫治疗后小鼠胃内H.pylori定植量改变,ELISA检测血清、胃和小肠样本中的特异性抗体水平变化,ELISA检测小鼠脾淋巴细胞IL-4和IFN-γ表达量的差异。【研究结果】 1.口服表达抗原表位融合蛋白CagA160-VacA62-UreB138 (CVU)的减毒沙门菌免疫治疗效果最好,清除率达62.5%,未清除小鼠胃内H.pylori定植量与空质粒菌组相比较显著降低。2.疫苗免疫后Th1应答与未免疫组相比较显著增强;疫苗免疫后H.pylori特异性抗体应答与未免疫组相比较显著增强。【结论】本研究结果表明:减毒沙门菌载体疫苗对H.pylori感染具有显著的免疫清除作用,免疫保护效果与特异性CD4 + T细胞Th1型应答、血清IgG和粘膜sIgA抗体的应答相关,且其疗效与融合表位蛋白的排列顺序有关。表达的抗原表位融合蛋白CVU的减毒沙门菌载体疫苗,疗效优于其他排列方式,可能成为抗H.pylori的候选疫苗。
[Abstract]:Helicobacter pylori ( H . pylori ) is a kind of spiral gram - negative bacteria implanted in human gastric and duodenal mucosa , which mainly causes chronic gastritis , peptic ulcer and gastric cancer .

Immunotherapy has been widely used internationally for H . pylori infection . The results of animal and human trials have shown that immunotherapy has the effect of removing H . pylori . It is suggested that the therapeutic vaccine has serious consequences for the treatment of H . pylori infection .

Conclusion : High levels of Th1 - biased response is necessary to protect against H . pylori infection and to induce mucosal immune response . Most studies have shown that high level Th1 - biased response is necessary to protect against H . pylori infection .

The related study shows that the oral immunization recombinant Salmonella vaccine has the characteristics of good safety and good immunogenicity . The full - length H . pylori protein molecule can be used as vaccine antigen to contain non - essential antigenic epitopes , and has a large molecular weight , is difficult to express and purify , and the fusion epitope antigen is easy to express and purify .

Purpose of research

1 . To evaluate the therapeutic effect of attenuated Salmonella carrier vaccine expressing different epitope fusion proteins in H . pylori infected mouse model .

2 . The role of Th1 and Th2 cell responses in the immune clearance of H . pylori infection was preliminarily defined .

Methodology of research

1 . Construction of the attenuated Salmonella carrier vaccine expressing H . pylori in different sequence of the epitope - fusion protein of A , VacA and UreB antigen , and evaluated its efficacy in mice model with H . pylori infection .

2 . Real - time PCR was used to detect the change of H . pylori colonization in the stomach of mice . ELISA was used to detect the level of specific antibody in serum , gastric and small intestine samples , and ELISA was used to detect the difference of IL - 4 and IFN - 纬 expression in spleen lymphocytes of mice .

Outcome of the study

1 . The results showed that attenuated Salmonella typhimurium vector vaccine had a significant effect on H . pylori infection . The results showed that attenuated Salmonella carrier vaccine had a significant effect on H . pylori infection . The results showed that attenuated Salmonella carrier vaccine had a significant effect on H . pylori infection , and its efficacy was related to the sequence of specific CD4 + T cell Th1 type response , serum IgG and sIgA antibody .
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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