壳聚糖-枸杞多糖微粒幽门螺旋杆菌疫苗Hp-WAE的治疗作用及机制研究

Current therapies for Helicobacter pylori (Hp) infection, based on a combination of three or four different antibiotics, entail problems such as increasing antibiotic resistance and re-infection. Therefore, there is an urgent task for development of therapeutic vaccine against Hp. In our previous studies, a variety of epitope vaccines based on Hp urease were constructed. The CTB-UE vaccine could reduce Hp colonization number in stomach of mice.However, as a therapeutic vaccine, the treatment effect of these vaccines needed to be improved and it lacked an oral delivery system. In this study, we constructed a therapeutic recombinant epitope vaccine Hp-WAE composed B and Th cell epitopes from many key Hp virulence factors and colonization factors on the basis of our previous research works by bioinformatics application software according to the empirical theories on vaccine construction, which may induce antibody-mediated humoral immune responses and cellular immune response with epitope specifity against many Hp antigens. A self-designed oral delivery system with chitosan and Lycium barbarum polysaccharides (LBP) microparticulates will be used to assist the Hp-WAE vaccine to treat Mongolian gerbil model infected with Hp. First, the immunogenicity and immunological specificity of Chitosan-LBP microparticles vaccine Hp-WAE will be evaluated by immunological and pharmacological methods. Lastly, the therapeutic effect and mechanism of Chitosan-LBP microparticles vaccine Hp-WAE will be elucidated. The study of Chitosan-LBP microparticles vaccine Hp-WAE is aiming to provide experimental and theoretical basis for the development of a therapeutic vaccine against Hp with independent intellectual property right.

多联抗生素疗法治疗幽门螺旋杆菌(Hp)感染性胃病面临着Hp耐药性、副作用和治疗后再感染等问题，因此研制治疗性Hp疫苗是一项重要而紧迫的课题。申请人前期基于Hp尿素酶构建了多种单价Hp表位疫苗，它们可显著地降低小鼠胃部Hp的定植数，减轻胃部炎症反应，但作为治疗性疫苗，疗效有待进一步提高。本项目在前期研究基础上，选择Hp多种关键毒力因子和黏附因子的B和Th优势抗原表位，通过表位疫苗的构建理论和生物信息学的分析，构建了一种具有科学合理结构、可激发高特异性的抗体体液免疫应答和混合型细胞免疫应答的多价表位疫苗Hp-WAE,现拟将其辅以自主设计的壳聚糖-枸杞多糖微粒口服疫苗投递系统，免疫治疗Hp感染的蒙古沙鼠模型，利用免疫学和药理学等方法从体内外分析多糖微粒疫苗Hp-WAE的免疫原性和免疫特异性，阐明其治疗作用及分子免疫学机制。本项目为我国研制出具有自主知识产权的治疗性Hp疫苗奠定理论依据和实验基础。

本项目按照研究计划顺利完成了各项研究内容，主要概述如下：在前期幽门螺旋杆菌（Hp）尿素酶表位疫苗（CTB-UE、CTB-UA、rCtUBE）的研究基础上，选择多种Hp关键毒力和黏附因子（UreA、UreB、HpaA、CagA、CagL、KatA、NAP、Lpp20、HSP60）的B和Th表位，通过表位疫苗的构建理论和生物信息学的分析，构建了多种可激发高特异性抗体体液免疫和细胞免疫应答的多价表位疫苗CWAE（Hp-WAE）、CFAdE和FVpE；利用基因合成、PCR和酶切偶联等分子克隆技术，成功构建了分别含有融合基因CWAE、CFAdE和FVpE的重组表达载体pETCWAE、pETCFAdE和pETFVpE，通过E. coli表达重组疫苗蛋白CWAE、CFAdE和FVpE，利用镍离子亲和层析和离子交换层析等蛋白纯化技术获得了高纯度重组蛋白CWAE、CFAdE和FVpE；然后，利用新鲜培养的国际标准菌株Hp SS1建立Hp感染的蒙古沙鼠，通过制备的壳聚糖--枸杞多糖（Chitosan-LBP）微球疫苗CWAE、CFAdE和FVpE免疫治疗患病小鼠，利用Hp定量培养、qPCR、尿素酶活性检测、组织病理学等方法评价多糖微球疫苗CWAE、CFAdE和FVpE治疗Hp感染的效果。实验结果证实多糖微球疫苗CWAE、CFAdE和FVpE具有较好的治疗Hp感染的效果；其次，利用ELISA、GM1-ELISA、Western blot、尿素酶活性中和等实验，分析多糖微粒疫苗CWAE、CFAdE和FVpE的免疫原性、特异性及其激发中和抗体的能力，检测免疫后沙鼠体内免疫淋巴细胞、抗体、细胞因子的种类、水平及变化，阐述多糖微粒疫苗CWAE（Hp-WAE）、CFAdE和FVpE的免疫保护性机制。实验证实多糖微粒疫苗CWAE、CFAdE和FVpE能够激发机体产生针对Hp多种黏附和毒力因子的高特异性IgG、IgA和sIgA抗体体液免疫应答和Th1/Th2/Th17混合型细胞免疫应答。本项目所设计的多糖微球Hp多价表位疫苗，结构全新，不易与其他Hp疫苗相关国际专利发生冲突，属于具有自主知识产权的候选生物技术药物，以项目负责人为第一或通讯作者发表论文5篇，其中SCI收录4篇，申请发明专利5项，获得宁夏科学技术进步奖三等奖1项，临床应用及成果转化潜力较大，具有巨大的社会和经济效益。