肺炎链球菌疫苗SPY1的一种免疫保护机制：TGF-β信号通路介导Treg细胞参与保护性免疫

Vaccine effectiveness depends on the mechanism of immunizing protection elicited; therefore, claiming the protective mechanism is a key part of development of novel vaccine candidate. Pneumococcal live attenuated vaccine SPY1 was an effective vaccine candidate developed before, showing characters of reduced virulence, high genetic stability, safety, and excellent protection in mice, which was even better than that of commercially available polysaccharide vaccine. Increase in Treg cell numbers in immunized mice was innovatively found when investigating the protective mechanism of SPY1. Further, we found elevated TGF-β concentration induced by SPY1, whereas, decrease in Treg cells and impaired protection were observed as results of inhibition of TGF-β. We therefore hypothesized that SPY1 is able to induce protective Treg immune response via TGF-β signal pathway, which participate in the protection of SPY1 via some immune modulating pathways. In this research, to further investigated the detailed signal pathways by which the protective Treg immune response elicited by SPY1, as well as the molecular mechanism of SPY1-specific Treg immune response maintaining the immune homeostasis and participating in the protection induced by SPY1, techniques including qRT-PCR, flow cytometry analysis, Western blot, and DNA microarrays for gene expression profiles will be employed. Our work will further provide theoretical and experimental evidences for further research of SPY1.

疫苗的有效性取决于其免疫保护机制，因此，阐明疫苗的免疫保护机制是新型疫苗临床前试验的关键内容。SPY1是前期研发的肺炎链球菌减毒活菌疫苗，该疫苗毒力显著降低，遗传稳定性高，安全性好，保护效果优于现有商品化多糖疫苗。前期探索SPY1免疫保护机制时，新发现SPY1可诱导小鼠Treg免疫反应；进一步发现免疫小鼠体内TGF-β1水平升高；抑制小鼠TGF-β，Treg细胞减少，疫苗保护效果降低。推测：SPY1是否通过TGF-β信号通路诱导Treg细胞分化，并通过免疫抑制性细胞因子或共刺激分子发挥免疫调节功能，参与疫苗保护效果？因此，本研究拟通过流式细胞术、功能分类表达谱芯片、qRT-PCR、Western blot等技术，深入探讨TGF-β信号通路介导SPY1诱导特异性Treg反应，发挥免疫调节功能，参与疫苗保护的机制，为阐明SPY1疫苗的保护效应机制提供理论和实验依据。

疫苗的有效性主要取决于其免疫保护机制，因此，阐明疫苗的保护机制是新型疫苗研发的关键部分。SPY1是本课题组前期研发的一株肺炎链球菌减毒活菌疫苗，该疫苗毒力显著降低，遗传稳定性高，安全性好，保护效果优于现有的商品化多糖疫苗。在之前的研究中，我们发现SPY1可诱导产生保护性的特异性Treg免疫反应，然而，其潜在机制并不清楚。本研究发现Treg细胞抑制剂P17上调免疫细胞因子（IL-12p70、IFN-γ、IL-4、IL-5、IL-17A）和炎症相关细胞因子TNF-α的水平，下调免疫调控因子IL-10和免疫抑制因子IL-6的水平，破坏SPY1疫苗接种诱导的系统性保护性免疫应答平衡，损害SPY1诱导的对肺炎链球菌定植引起肺损伤的保护，感染小鼠的肺部组织损伤和炎症反应明显加重，从而消除SPY1对小鼠的免疫保护作用。SPY1免疫可诱导小鼠肺中Treg细胞显著增加，免疫小鼠肺中Treg细胞转录因子Foxp3的表达上调，并可被P17显著抑制，提示SPY1免疫激活了特异的获得性Foxp3表达。在SPY1免疫小鼠肺匀浆、脾匀浆和脾上清中，TGF-β1水平显著增加，在肺炎链球菌19F感染后，SPY1免疫对TGF-β1的上调可被P17处理逆转，证明SPY1免疫激活了TGF-β1的产生诱导获得性Foxp3的表达。肺炎链球菌19F感染后，PCR结果显示免疫组smad2、smad3和smad4 mRNA表达增加，smad7 mRNA表达降低，肺组织中上调的Smad2/3和p-Smad2/3以及下调的Smad7通过WB证实，且其变化趋势具有时间依赖性，证明SPY1免疫激活TGF-β1-Smad2/3信号通路参与特异性Treg细胞的产生。SPY1免疫组PD-1和CTLA-4的水平增加，并可被P17处理逆转，提示PD-1和CTLA-4参与SPY1特异性Treg细胞在疫苗中免疫保护作用。研究结果提示TGF-β1参与疫苗SPY1对肺炎链球菌定植引起肺损伤的保护，SPY1刺激的TGF-β1对SPY1诱导的系统性保护性免疫应答之间的平衡是必要的。激活的TGF-β1-Smad2/3信号通路参与了SPY1特异性Treg细胞的产生，Treg细胞通过PD-1和CTLA-4的表达增加参与疫苗的免疫保护作用。本研究内容的完成，进一步深入阐明了减毒活菌疫苗SPY1的保护效应机制，为该疫苗的临床前试验提供理论和实验依据。