脂阿拉伯甘露聚糖和荚膜阿拉伯甘露聚糖对卡介苗IgG Fc段糖基化修饰及功能的调控

The data from tuberculosis-infected population show that specific IgG may be used against M. tb, the specific Fc domain glycosylated patterns, including the high levels of digalactose and sialic acid, and less fucose contribute to bacterial control via Fc-mediated antibody effector functions. Because BCG provides protection to disseminated TB during early childhood, and all candidate vaccines against tuberculosis which aimed at enhancing cell immunity have not demonstrated more protective efficacy than BCG in humans. Therefore, our hypothesis is that the specific IgG and Fc glycosylated patterns may play a role in BCG induced protection which tuned by surface polysaccharides. We have detected sialylated and galactosylated modifications in PPD-specific IgG induced by BCG and established a set of methods to analyze Fc glycosylated patterns. Therefore, the following parts will be performed in this grant. 1. To further detect the patterns of Fc glycosylation in BCG-specific IgG and to determine the functions of glycosylated BCG-specific IgG. 2. To regulate the Fc glycosyation via lipoarabinomannan（LAM）and capsular arabinomannan (AM). Furthermore, a recombinant BCG with high level of LAM and AM will be constructed and investigated. 3. To explore the optimal glycosylated patterns of antimycobacterial vaccine by animal challenge model. The results we acquired will shed light on mechanisms of BCG-induced humoral immunity, and provide the theoretical basis of vaccine optimization.

结核感染人群的研究表明结核分枝杆菌特异性IgG在防御结核感染中发挥作用,尤其是其Fc段糖链的高双半乳糖、高唾液酸和低岩藻糖化，可提高巨噬细胞等Fc-FcγRs路经介导的杀菌效应并限制感染。因为卡介苗主要降低儿童播散性结核的发病率，而激活细胞免疫的结核候选疫苗保护力并未优于卡介苗。因此我们假设：卡介苗特异IgG及其Fc段糖基化发挥保护作用，并且部分抗原就是表面多糖。我们利用小鼠实验检测到卡介苗IgGFc段含有唾液酸和半乳糖，并初步建立了IgG糖基化修饰的解析方法。本项目将开展：1.分析卡介苗IgGFc段的糖基化修饰和功能；2.利用胞壁脂阿拉伯甘露聚糖、荚膜阿拉伯甘露聚糖以及重组卡介苗调控卡介苗IgGFc段的糖基化修饰；3.用细菌攻击实验等评价调控后卡介苗的抗结核免疫效果，发现有利于机体的糖基化修饰形式。本项目将从IgG糖基化修饰角度揭示卡介苗的体液免疫机制，为优化卡介苗提供理论基础。

BCG作为唯一许可预防结核病的疫苗，能降低儿童播散性结核的发病率。因此全面认识BCG的体液免疫保护机制对BCG优化具有启示作用。本项目主要聚焦于胞壁多糖结合物中的荚膜AM以及BCG分泌的甘露糖基化蛋白调控BCG特异IgG的研究。 （一）我们纯化并鉴定出BCG荚膜AM，在小鼠体内证实AM 属于Th1型免疫调节分子，能增强BCG诱导的迟发性超敏反应。同时还构建了一系列高丰度的荚膜AM/LAM的重组BCG（rBCG），并评价rBCG的生物学功能。（二）在BCG分泌的甘露糖基化蛋白方面，我们联合ConA-凝胶层析技术和质谱技术从BCG的培养滤出液中鉴定了15种甘露糖基化蛋白，并借助BCG多克隆抗体筛选出2种免疫优势抗原即BCG_0470和BCG_0980。然后利用体外表达纯化技术，证实这两种糖蛋白存在甘露糖基化修饰，其糖链链接方式为α-链接。最后开展动物实验证实免疫优势糖抗原BCG_0470和BCG_0980在体内通过诱导迟发性超敏反应来激活T细胞，促进Th1类细胞因子IFN-γ和TNF-α，IL-2分泌，而且BCG_0470的甘露聚糖链是潜在的B细胞抗原表位和诱导高水平特异IgG的关键成分。综上所述，团队完成了研究任务，发表SCI文章1篇、CSCD文章2篇，撰写SCI文章1篇和核心期刊文章1篇，培养临床医学“5+3”一体化学生1名。本研究提示胞壁多糖结合物中的AM和O-甘露糖基化蛋白质及其寡糖链修饰在BCG的体液免疫保护中发挥作用，提示可从细菌胞壁多糖结合物的调控角度来优化BCG。