The Role of HBeAg in HBV Persistence

摘要 慢性乙肝病毒(乙肝)感染仍然是一个严重的公共卫生负担，影响到大约240人 全世界有100万人，美国至少有120万人，在中国是地方病，那里有8%-9% 的人口被感染。慢性乙型肝炎(CHB)是肝硬变的主要危险因素之一。 致命的肝癌。因此，阐明乙肝病毒持续存在的机制并寻找治疗方法具有重要意义。 慢性乙型病毒性肝炎的发展是由于宿主免疫系统未能清除 病毒感染，以及持久形式的HBVDNA基因组的寿命，这是共价封闭的 核微染色体结构中的环状(CCC)DNA。在这个研究项目中，我们将着手阐明 未被充分研究的乙肝e抗原(HBeAg)在乙肝病毒持续存在中的作用。HBeAg阳性和高滴度 反映慢性乙型肝炎患者体内高水平的乙肝病毒复制和免疫耐受。HBeAg血清转换通常是 被认为是一个有益的里程碑，也是病毒复制减少的证据。我们的初步观察 结果显示：(1)外周血中单核细胞髓系来源的抑制细胞(MMDSCs)数量 免疫耐受的慢性乙型肝炎患者高于健康对照组和免疫活跃患者；HBeAg诱导 MMDSCs的扩增和免疫抑制分子的上调，包括吲哚胺2，3- MMDSCs中的二氧合酶(IDO)，进而抑制T细胞的增殖和干扰素-γ的产生，提示 HBeAg可能通过激活mMDSC诱导免疫耐受/抑制；(2)细胞内 HBeAg中间体(P22)，而不是上清液中成熟的HBeAg，抑制干扰素敏感的活性 干扰素-α作用下的反应元件和干扰素刺激基因的表达 刺激，提示p22可能钝化干扰素信号，帮助病毒逃避先天免疫反应和 对干扰素治疗产生抵抗力；(3)p22的核定位表明，与HBeAg相似 与ccccDNA结合的乙肝病毒核心蛋白，p22可能与ccccDNA相互作用 微染色体并调节其稳定性和/或转录。通过利用一组与乙肝病毒相关的 分子生物学、免疫学、生物化学和蛋白质组学技术，以及几种创新的细胞培养 为研究HBeAg和ccDNA而专门设计的工具，我们建议进一步阐明 HBeAg-mMDSC-IDO轴诱导的T细胞抑制及细胞内HBeAg介导的干扰素阻断 信号，以及核HBeAg与cccDNA及其功能的潜在联系将被确定和 与核心蛋白相比。该项目的完成将有助于阐明乙肝病毒的作用机制 持久性，并最终导致新疗法的发展，以打破病毒诱导的免疫 耐受性和重置/重新激活免疫系统，以清除乙肝病毒感染。

ABSTRACT Chronic hepatitis B virus (HBV) infection remains a significant public health burden affecting approximately 240 million individuals worldwide and at least 1.2 million in the United States, and is endemic in China where 8-9% of the populations are infected. Chronic hepatitis B (CHB) is one of the leading risk factors of cirrhosis and the deadly liver cancer. Therefore it is important to elucidate the mechanism of HBV persistence and find a cure for chronic hepatitis B. The development of HBV persistence is due to the failure of host immune system to clear the virus infection, and the longevity of the persistent form of HBV DNA genome, which is the covalently closed circular (ccc) DNA in a nuclear minichromosome structure. In this research project, we will set out to elucidate the role of the understudied hepatitis B e antigen (HBeAg) in HBV persistence. HBeAg positivity and high titer reflect the high level of HBV replication and immune tolerance in CHB patients. HBeAg seroconversion is usually considered to be a beneficial milestone and evidence of reduced viral replication. Our preliminary observations revealed the followings: (1) The numbers of circulating monocytic myeloid-derived suppressor cells (mMDSCs) in immune tolerant CHB patients are higher than healthy controls and immune active patients; HBeAg induces the expansion of mMDSCs and the upregulation of immune suppressor molecules including indoleamine 2,3- dioxynase (IDO) in mMDSCs, which in turn inhibit T cell proliferation and IFN-gamma production, suggesting that the HBeAg may induce immune tolerance/suppression through activation of mMDSC; (2) The intracellular HBeAg intermediate (p22), but not the supernatant mature HBeAg, inhibits the activity of interferon-sensitive response element (ISRE) and interferon-stimulated gene (ISG) expression under interferon-alpha (IFN-α) stimulation, suggesting that p22 may blunt IFN signaling to help the virus to evade innate immune response and become resistant to IFN therapy; (3) The nuclear localization of p22 indicates that, reminiscent of the HBeAg homologue HBV core protein which has been shown to bind cccDNA, the p22 may interact with cccDNA minichromosome and regulate its stability and/or transcription. By making use of a battery of HBV-related molecular biology, immunology, biochemistry, and proteomics technologies, and several innovative cell culture tools specially designed for HBeAg and cccDNA studies, we propose to further elucidate the mechanisms of the HBeAg-mMDSC-IDO axis-induced T cell suppression and the intracellular HBeAg-mediated blockage of IFN signaling, and the potential association of nuclear HBeAg with cccDNA and its function will be determined and compared to core protein. The accomplishment of this project will shed light on the mechanism of HBV persistence, and ultimately lead to the development of novel therapeutics to break the virus-induced immune tolerance and reset/reactivate the immune system to clear HBV infection.