表面展示M细胞和TLR5配体提高牛腺病毒肠道黏膜免疫作用研究

Gut mocusal immunity is the decisive factor for oral vaccine efficiency.Gastrointestinal stability and intestinal transduction efficiency plays important role on the gut mocusal immunity of adenovirus. In this project, we take bovine adenovirus type 3(BAdV-3) as example, and explore the other factors and potential strategies to enhance BAdV-3 gut mucosal immunity from the view of antigen sampling and innate immune pathways of adenovirus. Firstly,we will construct the recombinant BAdV-3(rBAdV3s) displaying M cell ligand (C terminal of Invasin,INV C) and TLR5 agonist(truncated flagellin,9Flg-85-111) on surface respectively or simultaneously. The result rBAdV3s are further identified by Western blot and Immunogold electron microscope.Secondly,the function of displayed ligands is identified by using the methods of luciferase activity,ileal loop experiment and detection model for TLR5 signal pathway.Finally,the influenza H5N1 HA(H5HA) is used as vaccine antigen,and the mouse are immunized orallly with H5N1 vacicne based on rBAdV3s vector displaying INV C and/or 9Flg-85-111 ligands and on control BAdV-3 vector without displaying above ligands,respectively. Through comparing the H5HA-specific cellular and humoral immune response in intestinal mucosa and peripheral blood,we can conclude whether M cell targeting or TLR5 signal pathway activation is the alternative strategy to elicit substantial gut mucosal immunity.Furthermore,whether there is a synergistic or combined effect between the two strategies can also be proved.The above research will further promote the study of safer and better oral vaccine based?on BAdV-3.

肠道黏膜免疫是决定口服疫苗免疫效果的关键，影响腺病毒肠道黏膜免疫主要因素为胃肠道稳定性和肠道转导效率。本项目以牛腺病毒（BAdV-3）为例，尝试从肠道抗原摄取及天然免疫两方面对影响腺病毒肠道黏膜免疫的因素及提高途径进一步探究。首先，构建表面展示小肠微皱细胞（M cell）靶向配体（INV C）和/或TLR5配体（9Flg-85-111）重组腺病毒（rBAdV3s），Western blot和免疫金电镜对重组病毒进行鉴定。其次，通过荧光素酶活性、回肠结扎实验和TLR5通路激活实验检测展示配体元件的功能。最后，以流感病毒HA为抗原，给小鼠分别口服免疫表面展示M细胞配体和/或TLR5配体的rBAdV3s与对照病毒，比较两者在黏膜及全身诱导特异性免疫水平的差异，验证M细胞靶向和TLR5通路激活是否可单独或联合提高BAdV-3肠道黏膜免疫，为下一步构建安全高效BAdV-3口服疫苗载体系统奠定基础。

肠道黏膜免疫是决定口服疫苗免疫效果的关键，影响腺病毒肠道黏膜免疫主要因素为胃肠道稳定性和肠道转导效率。本项目以牛腺病毒（BAdV-3）为例，尝试从肠道抗原摄取及天然免疫两方面对影响腺病毒肠道黏膜免疫的因素及提高途径进一步探究。首先，构建表面展示TLR5 配体9Flg-85-111和M细胞配体Co1的重组腺病毒（rBAdV3s），并证明表面展示TLR5 配体9Flg-85-111和M细胞配体Co1 可提高BAdV-3载体疫苗免疫效力。其次，尝试通过与猪肠道腺病毒PAdV-3 fiber 替换提高BAdV-3的肠道嗜性 ，发现PAdV-3 fiber不能与BAdV-3成功替换。这些研究可为下一步构建安全高效BAdV-3口服疫苗载体系统奠定基础。