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

项目摘要 慢性B型肝炎（CH B）影响全球超过2.5亿人，每年约有100万人死于肝脏疾病。 疾病和肝细胞癌。高达28%的艾滋病毒感染者（PLWH）也患有慢性乙型肝炎。以来 HIV增加了CHB的肝病进展，因为肝病是CHB患者死亡的主要原因， 艾滋病毒感染者采取抗逆转录病毒治疗，开发一种HBV治愈方法势在必行。当前的核苷（酸）治疗 可以控制HBV复制，但不能治愈CHB，因为它不能根除稳定的共价闭合的 环状DNA（cccDNA），HBV复制的模板，来自肝细胞。此外，美国FDA定义 HBV治疗包括从血液中清除总的B表面抗原（tHBsAg）。这种简单的方法 tHBsAg来源的复杂性（来自cccDNA或HBV）使该定义不成立 整合到宿主基因组中的DNA（iDNA）。区分这两个来源的贡献， HBsAg对靶向治疗很重要。此外，我们使用液滴数字新技术的数据 PCR（ddPCR）证明NUC出乎意料地减少来自cccDNA的pgRNA的转录，但 编码tHBsAg的转录物S mRNA的转录是否也减少还不清楚。到 为了解决这些知识空白，我们建议从以下方面确定cccDNA和iDNA对tHBsAg的贡献： 69例患有不同阶段CHB的PLWH，其中60例进行了配对活检。在这些人的一个子集中，我们 将检查单个肝细胞，以确定具有iDNA和cccDNA的肝细胞的比例。 本研究中的69例患者（129例活检，因为60例患者进行了配对活检）具有不同阶段的HBV 感染包括免疫活性CH B（HBeAg+和HBeAg阴性）、非活性CH B和隐匿性肝炎B。要求1 将使用来自6名CHB患者的大块肝组织的RNA测序来构建表面（S）mRNA图谱， 将允许我们确定来源于cccDNA与iDNA的S mRNA的比例。后者 这是因为iDNA将在人类基因组中终止，在其3'端截短病毒序列。 然后，图谱将用于发现整合时发生的S mRNA中的主要断点， 开发了一种多重ddPCR，用于研究来自一个亚组的69个大块肝组织和单个肝细胞， 个体目标2将询问来自60个个体的肝活检，在此期间进行纵向活检， 时间他们对NUC了解NUC如何影响这些比例。目标1和2的数据将 与血浆定量HBsAg和大、中、小HBsAg的循环量相关。 我们还将确定CD4+ T细胞耗竭是否影响源自iDNA的HBsAg比例。 我们的研究将通过使用新技术来确定tHBsAg的比例， 从iDNA或cccDNA，这将为HBV治愈疗法的合理设计提供信息。

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.