Unraveling sources of hepatitis B surface antigen before and after nucleos(t)ide analogue treatment in People with HIV

揭示艾滋病毒感染者核（酸）类似物治疗前后乙型肝炎表面抗原的来源

基本信息

批准号：
10159638
负责人：
ASHWIN BALAGOPAL
金额：
$ 24.56万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-25 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10159638
关键词：
Address Affect Antigens Archives Biological Assay Biopsy Blood Cell surface Cells Cessation of life Chronic Chronic Hepatitis B Circular DNA Cirrhosis Clinical Complementary DNA Data Development Disease Progression Genes Genetic Transcription Genome Genomics HIV Hepatitis B Hepatitis B Surface Antigens Hepatitis B Virus Hepatitis B e Antigens Hepatocyte Human Genome Individual Infection Knowledge Length Liver Liver diseases Malignant neoplasm of liver Maps Measurement Measures Messenger RNA Outcome Persons Plasma Primary carcinoma of the liver cells Production Publishing Reporting Research Residual state Risk Sampling Source Specimen Strategic Planning Surface Surface Antigens Techniques Testing Time Tissues Transcript Transcriptional Regulation United States National Institutes of Health Viral Viral Genome Virion Virus Replication analog antiretroviral therapy base design digital genomic RNA innovation laser capture microdissection liver biopsy mortality novel nucleoside analog therapy design tool transcriptome sequencing viral DNA viral RNA virology

项目摘要

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 stop 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. The US FDA defines HBV cure has elimination of hepatitis B surface antigen (HBsAg) from blood. The simplicity of this definition is belied by the complexity of the source of HBsAg, 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 single cell laser capture microdissection (scLCM) and droplet digital PCR (ddPCR) demonstrate that NUCs unexpectedly decrease transcription of pgRNA from cccDNA but whether transcription of S is also reduced is unknown. To address these knowledge gaps, we propose to determine the contribution of cccDNA and iDNA to HBsAg at the single hepatocyte level before and during NUC in 5 HIV-HBV co-infected individuals with paired archived liver tissue. This proposal will use archived paired liver tissue from 5 HIV-HBV co-infected individuals of whom 2 were not and 3 were on NUCs at biopsy 1. At biopsy 2, obtained ~3 years later, all 5 individuals were on NUCs. Aim 1 will use RNA seq on bulk liver tissue from biopsy 1 to construct surface (S) transcript maps, which will allow us to determine the proportion of S transcripts that originate from cccDNA versus iDNA. The latter are distinguished because iDNA will terminate in the human genome. The maps will then be used to find major breakpoints in S that occur with integration allowing development of a multiplex ddPCR to study single hepatocytes. This multiplex ddPCR will be applied to 100-200 single hepatocytes dissected with scLCM to uniquely quantify the S transcripts from cccDNA versus iDNA in each hepatocyte from biopsy 1. Aim 2 will then utilize hepatocytes from biopsy 2 to understand how NUCs affect these proportions. Data from Aims 1 and 2 will be correlated with plasma quantitative HBsAg. We will also determine if NUCs affect global cccDNA transcription or only for pgRNA by combining these data with our prior data from the same hepatocytes. Our research will broadly impact the field by using novel techniques to determine the proportions of HBsAg from cccDNA or iDNA, which will inform the rational design of therapies for HBV cure. In addition, this proposal will determine whether NUCs can silence cccDNA globally or are only specific for pgRNA.

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。自从 HIV 会加速慢性乙型肝炎 (CHB) 的肝病进展，因为肝病是导致慢性乙型肝炎 (CHB) 死亡的主要原因对于接受抗逆转录病毒治疗的感染者来说，开发乙型肝炎治愈方法势在必行。目前的核（酸）苷（NUC）疗法可以阻止 HBV 复制，但不能治愈 CHB，因为它不能消除稳定的共价闭合来自肝细胞的环状 DNA (cccDNA)，是 HBV 复制的模板。美国 FDA 定义乙型肝炎治愈方法消除血液中的乙型肝炎表面抗原 (HBsAg)。这个定义的简单性被掩盖了 HBsAg 来源的复杂性，源自整合的 cccDNA 或 HBV DNA 进入宿主基因组（iDNA）。区分这两种来源对 HBsAg 的贡献对于目标是开发治疗方法。此外，我们的数据使用单细胞激光捕获新技术显微切割 (scLCM) 和液滴数字 PCR (ddPCR) 证明 NUC 意外减少 pgRNA 从 cccDNA 转录，但 S 的转录是否也减少尚不清楚。致地址这些知识差距，我们建议一次性确定 cccDNA 和 iDNA 对 HBsAg 的贡献 5 名 HIV-HBV 共感染者及其配对存档肝组织在 NUC 之前和期间的肝细胞水平。该提案将使用来自 5 名 HIV-HBV 共感染者的存档配对肝组织，其中 2 名未感染者其中 3 人在第 1 次活检时接受 NUC。在约 3 年后进行的第 2 次活检中，所有 5 人均接受 NUC。目标1 将使用来自活检 1 的大量肝脏组织的 RNA seq 来构建表面 (S) 转录图谱，这将使我们能够以确定源自 cccDNA 与 iDNA 的 S 转录本的比例。后者是之所以区分，是因为 iDNA 将终止于人类基因组中。然后，这些地图将用于寻找主要的 S 中的断点与集成一起发生，允许开发多重 ddPCR 来研究单个肝细胞。该多重 ddPCR 将应用于使用 scLCM 解剖的 100-200 个单个肝细胞，以独特地量化活检 1 中每个肝细胞中 cccDNA 与 iDNA 的 S 转录本。目标 2 将然后利用活检 2 中的肝细胞来了解 NUC 如何影响这些比例。目标 1 的数据和2将与血浆HBsAg定量相关。我们还将确定 NUC 是否影响全局 cccDNA 通过将这些数据与我们之前来自相同肝细胞的数据相结合，进行转录或仅针对 pgRNA。我们的研究将通过使用新技术来确定 HBsAg 的比例，从而对该领域产生广泛影响来自 cccDNA 或 iDNA，这将为乙肝治疗的合理设计提供信息。此外，本提案将确定 NUC 是否可以全局沉默 cccDNA 还是仅对 pgRNA 具有特异性。