新型复合疫苗佐剂高效诱导Th1/Th2免疫应答的作用机制研究

These adjuvant of FDA approved will limit widely applied in vaccine, because they can’t be stimulated efficiently the immune response of Th1 and Th2. The immune stimulatory molecules of Ophiopogon Saponins and MPLA, which induced efficiently the immune response of Th1 or Th2, are found by our large scale screening the animal or plant immune-stimulatory molecules. Base on the carriers with the double molecule of nanoemulsion, the novel combination adjuvant, which is inner layer of Ophiopogonin wrapped and the outer layer of MPLA adsorbed were papered, the carriers with the double molecule of nanoemulsion. Research suggests that the novel combination adjuvant has good quality and safety. And also, it has been confirmed that the novel adjuvant significantly enhance the immune response thorough inducing efficiently the immune response of Th1 and Th2. However, it is unclear that the specific immune response spectral characteristics and enhance the role of the mechanism. Thus, the differences in gene transcription sites, which causes immune enhancement was found by Gene chip, firstly. Then, the immune response of specific antibody and cytokine expression of differential expression profiles were detected by these methods including ELISA, ELISPOT and flow cytometry. At last, the immune enhancement mechanism of novel adjuvant were clarified by many methods such as real time fluorescence quantitative, immunohistochemistry, western blot and transcription set of chips, in these mouse model of MyD88 and NF-κB gene knockout. It will provide an importantly scientific basis and a solid technical support for the development of novel adjuvant field because the immune enhancement mechanism of novel adjuvant was explained.

现FDA批准佐剂都不能同时高效诱导Th1和Th2免疫应答，这极大限制疫苗的广泛应用。本课题组对多种天然动物植物免疫刺激分子的筛选，成功获得能高效诱导Th1或Th2的免疫刺激分子麦冬皂苷和MPLA。在此基础上，以纳米乳为载体，成功制备出质量稳定、安全性好的新型复合佐剂。经初步研究证实，该佐剂辅助MRSA疫苗可同时高效激发Th1和Th2免疫应答，具有明显免疫增强作用，但其具体免疫应答谱特征及增强作用机制尚不清楚。因此，本课题拟先通过基因芯片找到引起免疫增强作用的基因转录差异化位点；再用ELISA、ELISPOT和流式细胞术找到特异性抗体及细胞因子表达谱的表达差异；最后用MyD88、NF-κB基因敲除小鼠模型，用荧光定量、免疫组化、免疫印迹和转录组芯片等阐明其对免疫增强作用机制。本课题的完成，将为阐明新型复合佐剂的免疫增强效应机制提供重要科学依据，也为我国新型佐剂领域的发展提供坚实的技术支撑。

摘要：我们利用前期从大量的天然植物免疫刺激分子中首次成功筛选的麦冬皂苷和胞外免疫刺激MPLA。我们利用MRSA蛋白疫苗抗原，对其免疫活性和最佳免疫剂量进行再次验证和确定。复合疫苗佐剂的表征、稳定性尤其是体内外毒性是佐剂研究的关键环节，因此我们对其详细考察。该佐剂对机体的免疫应答水平、细胞因子种类以及疫苗维持时间，及可广泛使用多种疫苗类型及其增强免疫应答作用的机制等核心问题进行研究。确定麦冬皂苷的最佳剂量为30μg/只后，成功制备出剂良好质量特征和稳定性的新型纳米乳复合佐剂。研究发现，复合佐剂能显著降低其对标准L929细胞毒性和急性灌胃、肌肉和溶血毒性，极大提高其安全性。通过对其血清中抗体应答，细胞应答水平检测发现，该复合佐剂能够显著增强其体液Th1和粘膜免疫Th2应答，且主要是诱导偏向Th1应答水平，且为维持疫苗机体免疫应答作用时间至少半年。研究发现复合佐剂能诱导机体产生高水平细胞因子（IFN-γ，IL-12和IL-17）以及诱导活化DC细胞产生前炎症TFN-α，IL-1β和IL-6细胞因子高水平表达。同时，该佐剂可增强模式抗原OVA、MRSA和大肠杆菌融合蛋白和幽门螺杆菌疫苗和OVA肿瘤表位肽疫苗，发现均具有良好的增强免疫应答作用。复合佐剂可通过激活NOD样受体信号通路，诱导IFI44/NGP基因信号通路，增强趋化因子和细胞因子的表达，诱导机体产生高效的免疫应答，发挥增强免疫作用。本项目的完成具有极大的临床应用和实际价值，为后期疫苗佐剂研究奠定坚实的科学依据和理论基础。