抗肺孢子菌感染疫苗的新策略及其防御机制

The incidence of CD4+ T cells defect cases has been increasing rapidly following the spread of HIV/AIDS, increasing radiotherapy and chemotherapy to treat malignant neoplasms; organs transplant recipient and many other reasons. Therefore, the opportunistic fungal infections such as Pneumocystis were increasing obviously among the CD4+ T cells defect populations. But it is quite deficient that prevent beforehand and effective specific therapeutic for those infective diseases. This study in found with strong immunogenicity of pneumocystis multiple epitope protein expressed by p55 chimeric gene,and with gene transfer of IP-10 does augment clearance of infection in mice depleted of CD4+ T lymphocytes, to design a DNA prime/ rMVA boost vaccine strategy by prime muscle immune and rMVA and the AdIP-10 mucosal boost immune. The antibody responses to vaccine will be observed by detecting the anti-fungal carbohydrate or protein antibodies in serum and BAL. The cytokines such as interferon-gamma and interleukin-4, interleukin-17 will be assayed in lung tissue and regional lymph nodes of experimental mice. The goal of these studies will be to define the mechanism of host defense against Pneumocystis infection, explore the anti-infection factors and the immune pathway in the individual of the CD4+T lymphocyte defects, and to use this information to develop vaccine approaches optimal for immune deficiency host, as well as for other opportunistic fugal infections in the cell-immune defect population. Will also afford a convenient and reliable means for detection or diagnosis of pneumocystis infection or pneumocystis pneumonia.

伴随着各类致CD4+ T细胞缺陷因素的增多，肺孢子菌等机会性感染呈明显上升趋势。其防治策略为当前之急需。本课题以免疫机能低下宿主存在非CD4+ T细胞依赖抗感染途径为依据，以我们预实验证明转染IP-10可增强动物肺内免疫防御效应为基础，以我们创建并证明可诱导完全性保护免疫的肺孢子菌p55多表位嵌合基因为平台，设计了DNA 疫苗初次肌肉免疫/重组痘病毒疫苗加强黏膜免疫的异质性抗原提呈体系（DNA prime/rMVA boost）和用免疫佐剂(AdIP-10)强化肺部免疫的新策略，研究其在CD4+ T细胞缺陷动物体内抗肺孢子菌感染的免疫效应，揭示参与免疫应答的要素和免疫途径，阐明免疫防御的分子机制。研究结果将为CD4+ T细胞缺陷宿主抗肺孢子菌感染疫苗研究呈现破冰性进展，为其他机会感染性疾病防治策略的研究开辟令人振奋的新航线。其副产品将为肺孢子菌感染的检测提供更加方便有效的、无创伤性新方法。

本课题主要完成了如下三方面研究：.1.抗肺孢子菌感染疫苗的新策略及其防御机制方面的研究：①构建了肺孢子菌特异性多表位DNA疫苗（pIRES-hrGFP-1a/TAG）；验证了其真核细胞表达水平；研究了其在免疫抑制的肺孢子肺炎大鼠模型中免疫效应。结果显示pIRES-hrGFP-1a/TAG可诱导大鼠产生一定的细胞免疫和体液免疫，具有良好的抗肺孢子菌感染免疫保护效应。②通过构建表达γ-干扰素诱导蛋白10基因（IP-10）的重组腺病毒表达载体（pAdIP10）；转染HEK293细胞中进行病毒包装、扩增和鉴定，用重组腺病毒pAdIP10载体免疫动物。结果表明IP-10可以通过局部基因转染而传递，并可在肺组织中表达；IP-10基因转染能够刺激肺组织募集CD4+ T淋巴细胞和CD8+ T淋巴细胞；IP-10的基因转染能够刺激肺组织募集Tc1型的CD8+ T淋巴细胞。③构建了携带肺孢子菌p55多表位串联基因（p55TAG）的腺病毒重组载体，在鉴定其在真核细胞HEK293中的高效表达的基础上，用构建的p55多表位串联基因腺病毒载体疫苗免疫小鼠后，能诱导小鼠体内细胞和体液免疫反应发生，发挥加强局部抗感染免疫效应。.2.肺孢子菌基因检测方法的建立及其临床应用价值方面的研究：建立了检测肺孢子菌18S rRNA基因的普通PCR，real-time PCR，Nest PCR，LAMP 和实时荧光LAMP（real-time flourescence LAMP）等多种检测技术。经PCP模型动物实验证明，这些方法具有特异性好、敏感性强、程序简单等优点。应用上述方法对患者呼吸道分泌物进行检测，证明其具有很好的临床应用价值，特别适用于非创伤性标本的检测。.3.肺孢子菌感染与COPD等慢性肺疾病相关性的研究：应用LAMP等基因检测技术COPD等慢性肺部疾病患者的痰标本，发现该患者群的肺孢子菌基因阳性率高达60.30%；通过PCP大鼠模型也证明，肺孢子菌感染可诱导与COPD相关的病理、免疫细胞及细胞因子等反应。这些结果对深入研究COPD的发病机制及预防措施具有重要意义。.本项目资助发表论文24篇其中SCI收录9篇。并获得实用新型发明专利一项。研究成果申报2017年辽宁省科技进步二等奖，正在审理中。