植物源多糖及寡糖引发平衡的Th1/Th2类免疫反应机制研究

Immunomodulators have been widely used in vaccine and tumor therapy in recent years. To function as a good vaccine adjuvant, the immune stimulator needs to induce a balanced Th1/Th2 responses for vaccines to confer protective immunity against infectious diseases. The widely used aluminum-based adjuvants in prophylactic vaccines are effective in eliciting functional antibodies. It elicits Th2-skewed immune response to generate high level antibodies, but it is insufficient in stimulating cellular immune response or cytotoxic lymphocytes (CTL) which could be essential for disease prevention in some cases. Many plant-derived polysaccharides (PSs), specifically those derived from Chinese medicinal herbs, were reported to promote both humoral and cellular immunity. In addition to their good safety profiles for human use, some of these polysaccharides showed some promises as vaccine adjuvants in stimulation of a more balanced Th1/Th2 immune responses. In this proposal, we will embark on the mechanistic studies of these polysaccharides such as polysaccharides from Isatis indigotica root (IIP-2), Indian buead (PCP-1/PCP-2), various oligosaccharides which are more amenable for organic synthesis in the immune stimulation along with different vaccine antigens. Using Raw264.7 macrophages, we can evaluate the macrophage phagocytosis induced by polysaccharides using high content imaging analyzing system. In animal studies, total specific IgG levels, specific antibody subtypes and functional antibody titers, will be characterized using mouse models. Phenotypes in murine spleen cells from vaccinated mice were measured by flow cytometry to determine the cellular immune response. Cytokine profiles in plasma will be determined by Luminex. RT-PCR array or whole-mouse genome sequencing analysis will be performed using RNAs from the spleens of mice with outstanding performance of PSs as immune stimulators. These results from in vitro and in vivo analyses would shed lights on the mechanism for stimulation of a balanced Th1/Th2 immune response. Screening and characterization methods could be developed for assessment of potentials for plant-derived PSs to be used as vaccine adjuvants.

近年来免疫调节剂在疫苗和肿瘤治疗药物等方面得到了广泛的应用，疫苗引发的Th1/Th2类免疫应答类型是疫苗有效刺激机体适应性免疫反应的关键指标之一。在疫苗中，最为常用且十分有效的铝佐剂主要引发Th2优势应答，产生高效价的保护性抗体，但在激活CTL (cytotoxic T lymphocyte) 方面可能有些不足。前期研究发现多种植物源多糖具有显著的免疫促进作用，且能够诱导相对平衡的Th1/Th2类免疫反应。本项目拟以板蓝根多糖（IIP-2）、茯苓多糖（PCP-1/PCP-2）以及寡糖等作为研究对象，利用流式细胞技术，高内涵成像技术，RT-PCR技术及DNA芯片技术等，开展细胞及分子水平系列研究，阐释多糖分子的免疫促进作用及其诱导相对平衡的Th1/Th2类免疫应答的机制，为进一步优化新型预防性及突破免疫耐受的治疗性疫苗佐剂的筛选及理性设计提供理论基础。

天然多糖分子具有储量丰富、易于修饰、高生物兼容性及低毒性等诸多优点，并且其在免疫调节、抗肿瘤及抗氧化方面具有显著的效果，是一种理想的药物载体基质。本研究主要通过制备含植物源多糖的复合佐剂（如甘草多糖联用铝佐剂）或者纳米化佐剂（如含壳聚糖纳米颗粒的佐剂）来提高佐剂对免疫应答增强的效果，平衡Th1/Th2免疫反应，助力含植物源多糖新型佐剂的研发。结果发现枸杞多糖、当归多糖、甘草多糖及菊粉多糖联合铝佐剂对重组蛋白VZV gE显示出良好的佐剂活性，其中甘草多糖联合铝佐剂可提高 IgG2类抗体水平，促进脾淋巴细胞增殖和细胞因子IFN-γ以及IL-4的分泌，此外，还能提高CD3+CD4+和CD3+CD8+ T细胞的百分比。其次，我们综述了几种具有免疫调节特性的碳水化合物，包括已用于疫苗制剂中的壳聚糖，β-葡聚糖，甘露聚糖和皂苷。并对这些含碳水化合物的纳米颗粒进行的制备以及表征进行了描述，对其所产生的效力及其特征展开了讨论。总之，这些含碳水化合物的纳米颗粒佐剂的开发将促进不同类型疫苗的合理设计，并可能有助于控制当前的大流行和未来出现的传染病。. 此外，疫苗质量保证是确保疫苗安全性和有效性的关键部分，可最大效率地提高接种后的免疫保护作用，最大限度地降低免疫接种后的不良反应。为此我们对HPV多个型别（HPV6/11/16/18）以及HEV的单克隆抗体的结合活性、中和活性、构象敏感性等性质进行鉴定，筛选出可能识别不同免疫优势表位的单抗用于构建 HPV以及HEV疫苗的免疫化学质量分析平台。接着利用已经成熟的质量分析平台，对含防腐剂以及铝佐剂的HPV6/11/16/18/HEV p239抗原原液以及含铝佐剂的戊型肝炎疫苗产品进行了研究。差示扫描量热仪(DSC)以及酶联免疫吸附(ELISA)结果显示铝佐剂吸附后的HPV6/11/16/18/HEV p239抗原可降低硫柳汞所致的蛋白不稳定现象，具有一定稳定抗原的作用；热加速实验中HEV疫苗(益可宁®)可保证其抗原性与免疫原性，证实了脱冷链运输的可行性。这些发现有助于提高我们理解疫苗中抗原的特征和功能表征。