靶向增强抗原递呈作用的DNA疫苗预防种植体周围炎的机制研究

Peri-implantitis is one of the common complications after implanting, which is closely related to the infection of Porphyromonas gingivalis (P.gingivalis). Previous NSFC research has found that the DNA vaccine of rgpA is more effective than P.gingivalis vaccine to induce immune response. The vaccine contained rgpA or kgp is effective but the vaccine contained rgpB which does not have haemagglutinin-adhesin(HA) domain is invalid. Further, IL-4 could play a role in enhancing the antigenicity of vaccine. How to improve the targeting ability of vaccine and enhance the effectiveness are key points to develop vaccine..The HA of rgpA is the segment which is used to stick to host erythrocyte and obtain protoheme, that maybe HA is more effective than the other segments. we will design the vaccine contained the whole rgpA DNA sequence, rgpA HA sequence respectively, to study the antigennicity of rgpA HA.. microfold cell(M cell) is the key point of oral vaccine to induce effective immunoreaction, and its specific ligand Co1 could combine with C5a receptor which is known as a surface receptor of M cell, and the effectiveness of delivering antigen is improved. we plan to confirm that DNA vaccine combined with Co1 could improve the function of M cell，improving the immunoreaction further. .DC (dendritic cell) is imperative for antigen presentation, and the polypeptide (RVG) extracted from rabies virus glycoprotein could combine with the acetylcholine receptor of DC, enhancing the submission of antigen. We will construct plasmid combined with HA of rgpA and Co1, and mix with polypeptide to get DNA vaccine with more immunogenicity and ability of antigen presentation to prevent peri-implantitis. In our research, we will focus on DNA vaccine used to prevent peri-implantitis, and verify the immune effects through testing relevant surface markers, such like MHC Class II, CD40, CD80, CD86 by designing experiment both in vivo and vitro. .The results of this experiment will be importment and instructive for preventing implantitis and peridontitis. And this idea will provide a creative method for the research of vaccines for other diseases.

种植体周围炎的发生与牙龈卟啉单胞菌(Porphyromonas gingivalis，P.g)的感染密切相关。申请者已结题国自然项目研究了针对该病的疫苗，发现：rgpA DNA疫苗较灭活的P.g菌体能诱导更强免疫应答；rgpA、kgp DNA疫苗免疫作用强，而rgpB无效；联合应用白细胞介素4可增强rgpA DNA疫苗免疫效果。如何增强DNA疫苗靶向性、如何提高免疫效率是影响疫苗发展的关键。.本项目拟探讨这两个问题。rgpA凝集区是P.g的主要致病片段，故rgpA凝集区可能是免疫原性更强的片段；Co1可与M细胞表面特异性配体C5a结合，促进M细胞抗原传递；多肽（RVG-P）能与树突状细胞(DC)表面乙酰胆碱受体结合，促进DC抗原递呈效率。本项目拟构建包含rgpA凝集区和Co1重组质粒并与RVG-P混合，以期获得靶向性更强、递呈效率更高的疫苗，并探讨作用机制，对种植体周围炎的防御有重要意义。

研究发现种植体周围炎与牙周炎的发生与牙龈卟啉单胞菌(Porphyromonas gingivalis，P.gingivalis)的感染密切相关，针对此类感染的预防性治疗，课题组早期研究已验证精氨酸牙龈素rgpA DNA疫苗能诱导机体更强免疫应答。在早期P.gingivalis致病牙周炎研究中已有证实树突状细胞（dendritic cells, DCs）在P.gingivalis侵入龈下后具有吞噬细菌并分解递呈抗原激活下游免疫反应的作用。主要研究内容包括：1、筛选靶向性更强的致病基因片段构建基因疫苗，提升基因疫苗免疫原性；2、通过转录组学筛选树突状细胞在优化基因疫苗的作用下产生的关键因子并验证，选择正向增加的相关分子构建免疫佐剂，并明确机制；3、通过牙周炎及种植体周围炎动物模型验证不同基因疫苗在体内诱导免疫反应的保护效果。.通过研究得出结果：1、rgpA黏附结构域(haemagglutinin-adhesin ,HA)及其内部功能结构域HAR构建的基因疫苗较 rgpA 疫苗具有更强的免疫原性，HA及HAR疫苗诱导的IgG明显升高，同时纵向观察IgG2a具有升高趋势，4周时最显著，而IgG1趋势不显著，呈现Th1方向的免疫应答，且两者之间没有统计学差异（P>0.05）；2、将HAR过表达慢病毒与树突状细胞共培养，能有效诱导树突状细胞成熟，经细胞表面抗体流式检测CD80，CD83，CD86，HLA变化显著，正向偏移达90%以上。后经转录组学测序，DCs相关趋化因子及趋化因子受体，如CCL19，CCL17,CCR7，FOXO1等均有提高，倍数明显增加。3、构建CCL19-HAR基因疫苗，行体内实验验证CCL19作为疫苗佐剂具有诱导DCs体内成熟趋化的效果，提示分子佐剂可以有效的提升树突状细胞抗原摄取和递升的效果。4、micro CT、HE撇片等优化基因疫更能够更有效地预防P.gingivalis引起的牙槽骨丧失，且CCL19-HAR组的效果更佳。.本项目能够获得靶向性更强、递呈效率更高的基因疫苗，并探讨了其对树突状细胞影响的作用，对细菌感染性植体周围炎、牙周炎防御具有重要意义，对牙龈卟啉单胞菌感染相关的其实系统性疾病如阿尔茨海默症、慢性肾炎等有重要作用，