轮状病毒NSP4蛋白的粘膜免疫佐剂效应及其分子机制研究

The research of the TLR ligand molecules as novel adjuvant is an important direction for mucosal vaccine development. Literature reports and our preliminary exploration showed that the rotavirus nonstructural protein 4 (NSP4) improves antibody levels to model antigen via mucosa-administered in animal, and NSP4 may play some role in regulating immunity via the TLR/MyD88 pathways. However, the molecular mechanisms how NSP4 enhances immunity and induces the innate immune response are not clear. In the present study, we will evaluat the mucosal adjuvant effect of NSP4, concluding their time-response relationship and dose-response relationship in mice. On the base of this, the percentage of lymphocyte subgroups and the secretion of key cytokines were observed to analyze induced immune response types. With the tools including biochip, flow cytometry, gene-knockout mice, and signal molecule blockers, the cytokine expression profiles, membrane receptors and the downstream signal transduction molecules, NSP4-stimulated macrophage will be explored. Further, the adjuvant activity of NSP4 will be detected in the lack of the TLR receptor or under the condition of blocking some key signal molecules in mice, to clarify the mechanisms that NSP4 enhance immunity and induce innate immune response. This research not only contributes to understand the characteristics and mechanism involved in rotavirus NSP4 as an adjuvant, but also provides reference for mucosal adjuvant development.

以TLR配体分子为新型佐剂的研究是粘膜疫苗开发的重要方向。文献报道和申请者初步研究显示：轮状病毒NSP4经粘膜途径可以提高模式抗原在动物体内的抗体水平，并可能通过TLR/MyD88途径发挥其调节免疫的作用。然而NSP4增强免疫、诱导固有免疫应答的具体分子机制尚不清楚。本课题拟在小鼠模型评价NSP4粘膜佐剂效应及其时效、量效关系的基础上，通过观察淋巴细胞亚类构成比和关键细胞因子的分泌，分析NSP4所引起的应答类型；进一步借助生物芯片、流式细胞术、基因敲除小鼠和信号分子阻断剂等研究手段，解析NSP4诱导巨噬细胞的细胞因子表达谱、膜受体及其下游信号转导分子，并在小鼠体内验证关键信号分子的缺失或阻断在NSP4佐剂活性中的作用，阐明NSP4增强免疫，诱导固有免疫应答的分子机制。本研究的开展有助于深化对轮状病毒NSP4佐剂效应特征及机制的认识，对新型粘膜佐剂的研究开发也具有参考价值。

轮状病毒（RV）NSP4蛋白是具有肠毒素活性的多功能TLR配体分子，本研究首先通过选择重组表达RV NSP4的内质网膜外胞质区（NSP4-CF）活性区域片段验证了NSP4可通过粘膜免疫途径发挥增强系统与粘膜应答的佐剂效应：NSP4与模式抗原共免疫可显著提高小鼠血清抗体IgG、IgA 和粘膜SIgA的水平；细胞免疫应答方面则显示出Th1偏倚的增强效应。进一步探讨RV-NSP4佐剂效应的作用机制：NSP4作为一种病毒所表达的病原相关分子模式（PAMP），可以被巨噬细胞为代表的固有免疫细胞表面的模式识别受体（PRR）所识别，本研究用NSP4-CF体外作用于小鼠巨噬细胞RAW264.7，结果诱导了包括IL-6、TNF-α等炎性细胞因子的分泌。这种对巨噬细胞的免疫激活作用还表现为上调TLR2、TLR4、CD80/CD86等免疫分子的表达，研究结果提示NSP4的作用不仅是通过与巨噬细胞表面的TLR2或TLR4单一分子结合，而是可以同时作用于TLR2和TLR4，并可经非MyD88依赖的信号途径向下游转导活化转录因子NF-κB，且不同受体间可能存在胞内信号转导的交联现象，从而实现激活固有免疫细胞，增强免疫应答的佐剂效应。本研究揭示了RV NSP4作用的多功能性、复杂性与其作为新型粘膜免疫佐剂物质的潜在价值，为开发包括免疫佐剂在内的新型生物反应调节剂（BRM）提供了实验基础与研究思路。