Identifying the source of hepatitis B surface antigen in people with hepatitis B-HIV co-infection

项目摘要 慢性乙型肝炎(CHB)影响着全球超过2.5亿人，每年约有100万人死于肝脏 疾病和肝细胞癌。高达28%的艾滋病毒携带者(PLWH)也患有慢性乙肝。自.以来 艾滋病毒会增加慢性乙肝的肝病进展，因为肝病是#年死亡的主要原因。 在接受抗逆转录病毒治疗的同时，开发一种治疗乙肝的药物势在必行。当前的核(T)胺(NUC)疗法 能控制乙肝病毒复制但不能治愈慢性乙肝，因为它不能根除稳定的共价闭合 来自肝细胞的环状DNA(CcDNA)，是复制乙肝病毒的模板。此外，美国FDA定义 乙肝治疗可清除血液中的总乙肝表面抗原(THBs)。它的简单性 THBs Ag来源的复杂性与定义不符，tHBs Ag来自cccDNA或乙肝病毒 整合到宿主基因组中的DNA(IDNA)。区分这两个来源对 乙肝表面抗原对于靶向开发治疗方法很重要。此外，我们的数据使用了水滴数字的新技术 聚合酶链式反应(DdPCR)显示，nucs意外地减少了cccDNA中pgRNA的转录，但 目前尚不清楚编码乙肝表面抗原的转录本S的mRNAs转录是否也减少了。至 为了解决这些知识差距，我们建议确定cccDNA和IDNA对tHBs Ag的贡献 69例慢性乙型病毒性肝炎不同分期，其中60例进行了配对活检。在这些个体的一个子集中，我们 将对单个肝细胞进行检查，以确定肝细胞与IDNA和cccDNA的比例。 在这项建议中，69名患者(129例活检，因为60例已配对活检)具有不同阶段的乙肝病毒。 感染包括免疫活动性慢性乙肝(HBeAg+和HBeAg-neg)、非活动性慢性乙型肝炎和隐匿性乙型肝炎。目标1 将使用6例慢性乙肝患者的肝组织标本的rna seq构建表面(S)mrna图谱， 这将使我们能够确定来自CCcDNA和IDNA的S基因的比例。后者是 之所以与众不同，是因为IDNA将终止于人类基因组，在其3‘端截断病毒序列。 然后，这些图谱将被用来寻找S mRNAs中随着整合而发生的主要断点，从而 建立多重ddPCR方法研究69个大块肝组织和单个肝细胞 个人。目标2将询问60个人的肝活检，在此期间进行纵向活检 他们需要时间来了解核是如何影响这些比例的。来自目标1和目标2的数据将是 与血浆中定量的乙肝表面抗原和大、中、小乙肝表面抗原的循环量相关。 我们还将确定CD4+T细胞耗尽是否会影响IDNA产生的乙肝表面抗原的比例。 我们的研究将通过使用新技术来确定tHBs Ag的比例，从而广泛影响该领域 来自IDNA或cccDNA，这将为合理设计治疗乙肝的方法提供信息。

Project Summary Chronic hepatitis B (CHB) affects over 250 million people worldwide, with ~1 million annual deaths due to liver disease and hepatocellular carcinoma. Up to 28% of persons living with HIV (PLWH) also have CHB. Since HIV increases liver disease progression from CHB and because liver disease is a leading cause of mortality in PLWH taking antiretroviral therapy, developing a HBV cure is imperative. Current nucleos(t)ide (NUC) therapy can control HBV replication but cannot cure CHB because it does not eradicate the stable covalently closed circular DNA (cccDNA), the template for HBV replication, from the hepatocyte. In addition, the US FDA defines HBV cure has elimination of total hepatitis B surface antigen (tHBsAg) from blood. The simplicity of this definition is belied by the complexity of the source of tHBsAg, which derives from either the cccDNA or HBV DNA that is integrated into the host genome (iDNA). Distinguishing the contribution of these two sources to HBsAg is important to target developing a cure. Further, our data using the novel techniques of droplet digital PCR (ddPCR) demonstrate that NUCs unexpectedly decrease transcription of pgRNA from cccDNA, but whether transcription of S mRNAs, the transcripts that encode for tHBsAg, is also reduced is unknown. To address these knowledge gaps, we propose to determine the contribution of cccDNA and iDNA to tHBsAg from 69 PLWH with different stages of CHB of whom 60 have paired biopsies. In a subset of these individuals, we will examine single hepatocytes to determine the proportions of hepatocytes with iDNA and cccDNA. The 69 individuals (129 biopsies since 60 have paired biopsies) in this proposal having varying stages of HBV infection including immune active CHB (HBeAg+ and HBeAg neg), inactive CHB, and occult hepatitis B. Aim 1 will use RNA seq on bulk liver tissue from 6 individuals with CHB to construct surface (S) mRNA maps, which will allow us to determine the proportion of S mRNA that originate from cccDNA versus iDNA. The latter are distinguished because iDNA will terminate in the human genome, truncating the viral sequence at its 3’ end. The maps will then be used to find major breakpoints in S mRNAs that occur with integration, allowing development of a multiplex ddPCR to study 69 bulk liver tissues and single hepatocytes from a subset of individuals. Aim 2 will interrogate liver biopsies from the 60 individuals with longitudinal biopsies during which time they were on NUCs to understand how NUCs affect these proportions. Data from Aims 1 and 2 will be correlated with plasma quantitative HBsAg and with circulating amounts of Large, Medium, and Small HBsAg. We will also determine if CD4+ T cell depletion affects the proportion of HBsAg that derives from iDNA. Our research will broadly impact the field by using novel techniques to determine the proportions of tHBsAg from iDNA or cccDNA, which will inform the rational design of therapies for HBV cure.