HBV CpG岛甲基化及TLR3传导通路在HBsAg阳性母亲婴儿 乙肝疫苗无弱应答中的作用

The high rate of non-/hypo-response to hepatitis B vaccine in infants born to HBsAg-positive mothers was a major challenge to preventing hepatitis B infection. In order to understand the mechanism of infant’s non-/hypo-response to hepatitis B vaccine, we propose to conduct a prospective cohort study and an in vitro study to explore the effects of maternal HBV CpG island methylation status on Toll-like receptor 3 (TLR3) signal pathway function and its related cytokines. Three specific aims are: 1) to explore the impact of HBsAg-positive mothers’ HBV CpG island I,II, and III methylation on mothers’ and infants’ HBV infection status, as well as infants’ non-/hypo-response to hepatitis B vaccine; 2)to examine the impact of TLR3/TRIF/ NF-κB, IRF3 signal pathway on expression of DC and B cells as well as Th1/Th2 cytokines, and subsequently understand its role in non/hypo immune response to hepatitis B vaccine; 3) employing an in vitro study through stimulating PBMC by HBV with different methylation patterns and then by HBsAg to investigate how HBV methylation status and activate or deactivate TLR3 signal pathway to influence immune response. This study has the potential to discover novel mechanism underlying non-/hypo-response to hepatitis B vaccine and subsequently improve infant’s immune response to hepatitis B vaccine.

HBsAg阳性孕妇婴儿乙肝疫苗无弱应答率高，严重影响乙肝防制效果，但机制未明。本项目在既往研究基础上，从HBV生物环境因素和启动乙肝疫苗免疫应答可能相关的遗传因素入手，以前瞻性研究和体外实验结合，探讨HBV CpG岛甲基化及天然免疫受体TLR3信号通路及其联合作用对婴儿乙肝疫苗无弱应答的影响。①探索HBsAg阳性母亲HBV CpG岛Ⅰ、Ⅱ和Ⅲ甲基化对母儿HBV感染状态及婴儿乙肝疫苗无弱应答的影响；②了解新生儿及婴儿TLR3/TRIF/NF-κB、IRF3信号通路对DC、B细胞等免疫细胞及Th1/Th2细胞因子的影响及变化规律，探讨其降低无弱应答发生风险的作用；③体外实验模拟婴儿暴露情况，用不同CpG岛甲基化的HBV处理及HBsAg刺激PBMC，比较不同甲基化状态HBV及活化与阻断TLR3信号通路对HBsAg免疫应答的影响，初步阐明其作用机制及逆转无弱应答的可能性，为降低其发生率提供新思路。

目的 探讨HBsAg阳性母亲HBV DNA CpG岛甲基化及TLR3信号通路对婴儿乙肝疫苗免疫应答的影响。方法 在所收集的399例HBsAg阳性母亲婴儿基础上，对其中HBV DNA载量≥106 IU/ml，且满足后续实验所需血清量的母儿111对，进行乙肝疫苗无弱应答影响因素的巢式病例对照研究。采用亚硫酸氢盐测序法进行CpG岛甲基化的检测，免疫组化检测胎盘TLR3信号通路蛋白的表达水平。体外培养PBMC，根据分组情况给予不同HBV甲基化状态血清、TLR3活化剂/抑制剂及rHBsAg刺激，流式细胞术检测TLR3信号通路蛋白、T、B和DC细胞及细胞因子。结果 HBsAg阳性母亲HBV DNA CpG岛II 和岛III CG位点甲基化率分别为11.14%(981/8806)和7.90%(218/2758)。母亲HBV DNA CpG岛II和岛III位于P基因区的CG位点甲基化组的PBMC HBV cccDNA的水平低于未甲基化组(115.82 IU/ml vs 5566.90 IU/ml，Z=2.02，P=0.043；26.20 IU/ml vs 1330.80 IU/ml，Z=-1.99，P=0.046)；婴儿乙肝疫苗无/弱应答组岛III及仅岛II的P基因区CG位点甲基化率高于强应答组（10.51% vs 7.33%，χ2=5.56，P=0.018；17.92% vs 9.76%，χ2=24.11，P<0.001），胎盘TLR3蛋白表达水平明显低于强应答组(Z=-2.16，P=0.031)。体外实验HBV甲基化组CD4+T细胞、浆细胞、mDC及pDC细胞占淋巴细胞的比例略低于未甲基化组（P>0.05），HBV甲基化组TLR3活化时，浆细胞比例升高(t=2.83,P=0.047)。结论 HBsAg阳性母亲HBV DNA CpG岛II CG位点甲基化率高于岛III。HBV DNA CpG岛甲基化可能会抑制HBV复制。在乙肝疫苗免疫应答方面，HBV DNA CpG岛CG位点甲基化可能会减弱机体对乙肝疫苗的免疫反应，胎盘TLR3蛋白则可能增强乙肝疫苗的免疫反应；当HBV甲基化存在时，活化TLR3可能会通过增加浆细胞的比例来降低HBV甲基化对乙肝疫苗免疫反应的减弱效应，为HBsAg阳性母亲婴儿这一特殊人群乙肝疫苗无/弱应答机制研究及降低其发生率提供了新思路。