Occult Hepatitis B Infection in South African HIV Patients

描述(申请人提供)：全球有3.5亿人慢性感染乙肝病毒(乙肝病毒)--这是世界上导致肝硬变和肝细胞癌的主要原因。慢性乙肝病毒感染的特征是血清中存在乙肝表面抗原。相比之下，隐匿性乙肝病毒感染(O-HBV)被定义为在没有可检测到的循环HBs Ag的情况下低水平的乙肝病毒复制。在人类和灵长类动物中，O-乙肝病毒的可传播性和随后慢性乙肝病毒感染的建立被很好地记录下来。此外，O-乙肝病毒与晚期肝纤维化、对干扰素治疗的反应性降低、肝细胞癌的发生和肝酶水平升高有关。在艾滋病毒阳性的队列中，O-乙肝病毒感染的流行率显著上升。在南非，艾滋病毒和乙肝病毒都是地方性疾病，艾滋病毒混合感染是O-乙肝病毒感染的主要风险因素。此外，乙肝病毒聚合酶(P)基因的耐药突变--它与表面(S)基因重叠--在南非很常见--甚至在接受治疗的人中也是如此--也可能影响乙肝表面抗原的表达。只有一小部分研究评估了这种缺乏体外乙肝表面抗原检测的潜在机制(S)。确定O-乙肝病毒感染特征的主要限制包括：1)缺乏对HBVDNA的灵敏定量分析；2)在具有复制能力的全长基因组的背景下，识别和表征与O-乙肝病毒相关的突变的能力有限。幸运的是，用于HBVDNA定量的高灵敏度、高成本效益的实时PCR分析--例如我们实验室开发的那种--现在已经可以使用。此外，还开发了一种新的方法，可以有效地扩增整个乙肝病毒基因组，也可以进行快速的功能分析。这项应用的目的是利用具有复制能力的全长乙肝病毒表达载体，确定O-乙肝病毒感染相关的S和P基因突变对肝细胞中乙肝表面抗原合成/保留/分泌和乙肝病毒复制的影响。对目前的乙肝筛查方法未能在体内检测到的O-乙肝病毒变异进行鉴定，并发展体外试验以评估缺乏乙肝表面抗原检测的机制(S)，将提高未来O-乙肝病毒感染的诊断敏感性，限制乙肝病毒的二次传播，为O-乙肝病毒的治疗方案提供关键信息，并提高我们对乙肝病毒复制如何促进肝细胞癌发展的理解。 公共卫生相关性：慢性肝病是艾滋病毒阳性患者发病和死亡的主要原因。在南非，乙肝病毒和艾滋病病毒都是地方病，艾滋病病毒是隐匿性乙肝病毒的一个重要风险因素--定义为在没有血清乙肝病毒表面抗原检测的情况下可检测到的HBVDNA。拟议的研究将探讨在这种地方性环境中缺乏表面抗原检测的流行病学特征和病毒学机制，并为未来对隐匿性乙肝病毒感染的体内和体外研究提供一个框架，最终可能导致提高对乙肝病毒感染的诊断敏感性，并限制继发感染。

DESCRIPTION (provided by applicant): Globally, 350 million people are chronically infected with hepatitis B virus (HBV) - the world's leading cause of cirrhosis and hepatocellular carcinoma (HCC). Chronic HBV infection is characterized by the presence of hepatitis B surface antigen (HBsAg) in the serum. In contrast, occult HBV infection (O-HBV) is defined as low level HBV replication in the absence of detectable circulating HBsAg. The transmissibility of O-HBV and the subsequent establishment of chronic HBV infection are well documented in humans and in primates. Moreover, O-HBV is associated with advanced liver fibrosis, reduced response to interferon therapy, the development of HCC, and increased liver enzyme levels. In HIV-positive cohorts, the prevalence of O-HBV infection is significantly elevated. In South Africa, both HIV and HBV are endemic, and HIV co-infection is a major risk factor for O-HBV infection. Moreover, treatment resistance mutations in the HBV Polymerase (P) gene - which overlaps with the Surface (S) gene - are common in South Africa - even among treatment-naove individuals - and may also impact HBsAg expression. Only a small number of studies evaluated the potential mechanism(s) for this lack of HBsAg detection in vitro. Major limitations to characterizing O-HBV infection include 1) the lack of sensitive quantitative assays for HBV DNA, and 2) the limited ability to identify and characterize mutations that are associated with O-HBV in the context of full-length, replication-competent genomes. Fortunately, highly sensitive, cost effective real-time PCR assays for HBV DNA quantification - such as that developed within our lab - are now available. Moreover, a novel method for efficient amplification of whole HBV genomes that also permits rapid functional analysis has been developed. The aims of this application are to determine the effects of S and P gene mutations associated with O-HBV infection on HBsAg synthesis/retention/secretion and HBV replication in hepatocytes using full-length, replication-competent HBV expression vectors. The identification and characterization of O-HBV mutations in vivo that are not detected using current HBV screening assays and the development of in vitro assays to evaluate the mechanism(s) underlying the lack of HBsAg detection would improve future diagnostic sensitivity for O-HBV infection, limit secondary transmission of HBV, provide critical information on treatment options for O-HBV, and improve our understanding of how HBV replication contributes to the development of HCC. PUBLIC HEALTH RELEVANCE: Chronic liver disease is a major cause of morbidity and mortality in HIV-positive individuals. In South Africa, where both HBV and HIV are endemic, HIV is a significant risk factor for occult HBV - defined as detectable HBV DNA in the absence of serum HBV surface antigen detection. The proposed studies will examine the epidemiologic characteristics and virologic mechanisms for this lack of surface antigen detection in this endemic setting and provide a framework for future in vivo and in vitro studies of occult HBV infection that could ultimately lead to improved diagnostic sensitivity for HBV infection and limit secondary infections.