靶向鸡DEC-205球虫病疫苗激发的保护性细胞免疫应答机制研究

Eimeria spp. are the most common protozoan pathogens of chickens. Infection by Eimeria causes coccidiosis which leads to signicant economic losses in the poultry industry. Fortunately, Eimeria infection can be effectively controlled by vaccination with live Eimeria oocysts. But this kind of vaccine has not been widely used due to the difficult preparation, and high price. The mechanism of the protective immunity elicited by eimeria infection need to be illuminated for the novel vaccine development. Chicken coccidia can not infect dendritic cells (DCs), but infection of chicken by this parasite can elicit solid cell mediated immunity. This phenomenon suggest that cross-presentation may exist in chicken. DC receptor DEC-205 plays a critical role in antigen cross-presentation in mammal. But the function of DEC-205 in chicken need to be illuminated. In this study, we will construct a novel recombinant subunit vaccine fusion protein using EtIMP1 fused with a single chain antibody fragment(scFv) against chicken DEC-205. SPF chicken will be immunized to evaluate the specific cell mediated immune responses induced by scFvCD205-EtIMP1. The clinical effects (intestinal lesion scores, body weights gain, and oocysts shedding) of chickens after E. tenella challenge will be measured to evaluate the protection level of the recombinant vaccine. In addition, a preliminary study about mechanism of the protective cell mediated immunity elicited by scFvCD205-EtIMP1 will be studied. Our research will provide some useful information for the development of coccidiosis vaccines.

鸡球虫病是危害养禽业最重要的原虫病，疫苗免疫被认为是该病防治的重要途径。现有活卵囊疫苗存在着各种各样的问题，新型疫苗的研究需要揭示清楚鸡抗球虫免疫的机理。鸡球虫不感染树突状细胞，却可激发较好的细胞免疫应答，提示了交叉递呈的存在。在哺乳动物上，DC表面受体DEC-205可介导抗原的交叉递呈。鸡EDC-205是否具有类似的功能，可以介导球虫抗原的交叉递呈，进而激发机体产生抗球虫感染的细胞免疫应答？为回答这一科学问题，本申请将利用鸡DEC-205的单链抗体，与EtIMP1C蛋白融合表达，构建可以靶向鸡DC细胞的重组蛋白疫苗。通过动物免疫实验，研究其激发的细胞免疫应答，并对免疫鸡攻虫，从鸡的体增重，肠道病变计分以及卵囊排出量方面对该重组疫苗激发的免疫保护效果进行评价。同时，对该疫苗靶向树突状细胞增强抗原交叉递呈的作用和机制进行研究，研究结果将有助于球虫病的免疫预防和疫苗开发。

鸡球虫病在世界范围内造成了重大的经济损失。疫苗接种是控制该病的重要手段，研究表明，将抗原靶向树突状细胞（DC）是增强对病原体免疫应答的一种有效的方法。在这项研究中，我们评估了由柔嫩艾美耳球虫免疫映射蛋白1（EtIMP1）和针对鸡DEC205（一种在DC和其他细胞表面表达的内吞受体）的单链抗体片段（scFv）组成的嵌合亚单位疫苗对柔嫩艾美耳感染的效力。首先，原核表达了scFvDEC205-1、scFvDEC205-2、EtIMP1C、scFvDEC205-1-EtIMP1C、scFvDEC205-2-EtIMP1C等多种蛋白并对蛋白的大小和纯度利用SDS-PAGE和WB进行了鉴定；然后免疫鸡，分析了这些蛋白激发的细胞免疫和体液免疫，并评价了这些亚单位疫苗对鸡球虫感染的免疫保护作用。结果表明，与仅用重组scFvDEC205、EtIMP1C、空白组相比，用融合蛋白scFvDEC205-1-EtIMP1C、scFvDEC205-2-EtIMP1C免疫鸡可以产生更强的IgG、IgA应答和IL-2、IL-12、IL-4和IFN-γ的分泌。与未免疫的攻虫对照组相比，它还可以改善体重增加、减少盲肠病理和减少卵囊排出量。在和DC细胞结合和刺激DC细胞成熟方面，通过间接免疫荧光、酶联免疫吸附、Western Blot法证明重组蛋白scFvDEC205-1、scFvDEC205-2、scFvDEC205-1-EtIMP1C、scFvDEC205-2-EtIMP1C具有能够与未成熟鸡骨髓源树突状细胞（chBM-DCs）表面DEC205结合的靶向作用。重组融合蛋白在与未成熟chBM-DCs细胞共孵育后细胞表面形态学发生变化，细胞吞噬能力减弱; 细胞表面分子CD11c、CD86荧光强度增强; RT-qPCR结果显示经LPS或目的蛋白孵育24 h后的DC Th1/Th2型细胞因子、MyD88相关信号通路分子mRNA表达量上调，说明scFvDEC205在不过分降低DC吞噬能力的情况下还能具有刺激未成熟chBM-DCs发育成熟的潜质，且能更好的将抗原靶向DC细胞，同时可以调节Th1/Th2平衡，维持机体稳态。融合蛋白pET28a-scFvDEC205-1-EtIMP1C、pET28a-scFvDEC205-2-EtIMP1C具有良好的抗球虫保护效果，为今后开发靶向DC的鸡球虫病疫苗给予了有力的支持。