肠毒素佐剂联合细粒棘球绦虫EgM123诱导肠道黏膜Mφ和DCs分化机制及抗虫效果的研究

The prevalence of cystic chinococcosis is high in western China, and dog plays a povital role in the transmission of the disease. Dog vaccination blocking the transmission of the parasite has been suggested an ideal for controlling echinococcosis. However, successfully and effectively activating the intestinal mucosal immune of dog is essential for developing a dog vaccine against Echinococcus granulosus, the pathogen of cystic echinococcosis. We have isolated and proved that EgM123 mixed by QuilA can partially protect dogs against E. granulosus challenge infection, but the intestinal mucosal immune response including activation of Mφ and DCs was not activated effectively. It is not known whether EgM123 combined with enterotoxin adjuvant LTB and CTB can activate intestinal mucosal immunity and improve immune protection. We assume that the optimization of EgM123 combined with adjuvant immunization program can activate intestinal mucosa Mφ and DCs, then induce CD4 cells to Th1 polarization, that promotes anti- E. granulosus resistance. In this study, we will fuse or mix EgM123 with LTB and CTB in different immunization routes to vaccinate mice, and identify Mφ differentiation and DCs maturation in both in vitro and in vivo assays. We will identify the local immune responses of intestinal tissues and systematic immune responses including CD4 T cells and serological changes by using techniques such as flow cytometry, western blot, qRT-PCR and immunohistochemistry. Dogs will be vaccinated and challenge-infected with optioned formation and route of vaccination to validate the protective effect. We will explore the way to activate intestinal mucosa Mφ, DCs and improve the anti-worm protection effect of dogs by optimizing the combination of subunit vaccines, adjuvant and immunization routes, and provide a scientific basis for developing dog vaccine stopping the transmission of echinococcosis.

包虫病在我国西部高发，犬是最重要传染源。犬疫苗阻断病原传播是急需的控制措施，有效刺激肠黏膜免疫则是疫苗研制成功的关键。我们已分离并证明EgM123与QuilA混合接种犬可取得一定保护，但肠黏膜免疫并未有效激活。能否联合肠毒素佐剂LTB和CTB，有效激活肠黏膜免疫，提高免疫保护尚不清楚。本研究假设优化的佐剂联合EgM123免疫方案可以激活肠道黏膜Mφ和DCs，继而诱导CD4细胞向Th1极化，促进抗虫效果。本研究拟通过EgM123与LTB、CTB融合或混合开展体外刺激实验及不同途径免疫小鼠，用FCM、WB、qRT-PCR及免疫组化等技术测定肠黏膜组织结构、免疫学改变、Mφ和DCs分化状态和Th1极化效应。选择最优方案接种犬，验证犬抗细粒棘球绦虫的保护效果。本研究通过优化佐剂与亚单位疫苗免疫方案，探讨激活肠黏膜Mφ和DCs提高犬抗虫保护效果的途径，为研制控制包虫病的犬用疫苗提供科学依据。

包虫病在我国西部高发，犬疫苗阻断病原传播是急需的控制措施，有效刺激肠黏膜免疫清除虫体或抑制虫体发育是疫苗研制成功的关键。本研究通过使用前期分离到的细粒棘球绦虫成虫高表达的EgM123蛋白，并与两种肠毒素佐剂霍乱肠毒素亚单位B（CTB）和大肠杆菌肠毒素亚单位B（LTB）分别通过偶联原核表达或表达后混合的方式，通过开展体内外实验来比较和分析对局部和系统免疫带来的改变，以及对实验动物肠道抑虫效果的影响，为制备犬抗棘球绦虫疫苗提供理论依据。.通过项目的实施发现，CTB或LTB与EgM123（EgM9）蛋白混合后免疫犬与EgM123（EgM9）偶联LTB或CTB免疫犬，两种方式均能使目的蛋白抗体滴度在不同时间有效升高，但二者无差别。两种佐剂与EgM蛋白联合也尚不能有效控制犬肠道荷虫数量。犬肠道组织学染色结果发现无虫组犬PP结（PPJ）和PP结旁（PPW）肠道炎症反应均较虫多组重。qPCR检测结果发现PPJ中细胞因子IL6、TCR-V、 IL13、ARG，及PPW中的IL17与荷虫量显著相关。初步表明可能较强的炎症反应有助于终末宿主的持续抑虫。棘球绦虫原头蚴经体外培养发现其生存与培养液中胰蛋白浓度密切相关，为此我们原核表达和纯化了幼虫阶段高表达的胰蛋白酶抑制剂（Serpin7244和1931）。同时通过制备兔源Serpin多克隆抗体，并加入终浓度为2%胰酶的培养基，发现其能明显提高Eg幼虫的生存状态。在体内外实验还发现原头蚴头节外翻，成虫性状发育与环境菌群构成密切相关。通过评价细粒棘球绦虫（Eg）和多房棘球绦虫（Em）两型包虫在小鼠体内交叉免疫保护效果，证明了免疫中间宿主Eg和Em粗抗原疫苗抗原性存在交叉，要实现彻底宿主Em虫体清除效果的疫苗抗原还需要来源于同种。以上研究为抗虫疫苗的研制提供了新的思路和关键点，对开拓新的抗虫疫苗研制方法具有理论价值和潜在的应用前景。