Characterization of silently HBV-infected hepatocytes in HIV co-infection

HIV 合并感染中 HBV 沉默感染肝细胞的特征

基本信息

批准号：
10215496
负责人：
ASHWIN BALAGOPAL
金额：
$ 77.94万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-14 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10215496
关键词：
Address Affect Antigens Archives CD4 Positive T Lymphocytes CXCL10 gene Cell Count Cells Cessation of life Chronic Hepatitis B Circular DNA DNA Markers Data Defective Viruses Demographic Factors Disease Progression Epigenetic Process Equilibrium Future Gene Expression Gene Silencing Genes Genetic Transcription Genome HIV HIV-1 HepG2 Hepatitis B Surface Antigens Hepatitis B Virus Hepatitis B e Antigens Hepatocyte Human Human immunodeficiency virus test Immune Immune System Diseases Immunologic Markers In Vitro Individual Interferon Type II Interferons Interleukin-6 Interruption Lead Linear Models Linear Regressions Liver Liver diseases Malignant neoplasm of liver Measures Medicine Messenger RNA Methods MicroRNAs Morbidity - disease rate Persons Primary carcinoma of the liver cells Process RNA Recovery Research Residual state Reverse Transcriptase Polymerase Chain Reaction Sampling T-Cell Depletion TNF gene Testing Time Tissue Banks Tissues Viral Viral reservoir Viremia Virion Virus Virus Replication antiretroviral therapy bisulfite co-infection cohort design digital epigenetic silencing experience experimental study genomic RNA immune activation in vivo innovation laser capture microdissection liver biopsy mortality multilevel analysis novel marker reconstitution viral DNA virus core virus genetics

项目摘要

Chronic hepatitis B (CHB) affects nearly 400 million people worldwide, with ~1 million annual deaths due to liver disease and hepatocellular carcinoma. HIV-1/hepatitis B virus (HBV) co-infection is common. HIV-1 increases liver disease progression from CHB with liver disease being a leading cause of mortality in HIV-1 infected persons taking antiretroviral therapy (ART). Thus, a cure for CHB is needed. Although dually active ART (DAART) controls HBV and HIV-1 viremia, it cannot cure CHB because it does not eradicate the stable covalently closed circular DNA (cccDNA) form of HBV, the template for replication, from the hepatocyte. Our preliminary data demonstrate that some hepatocytes contain cccDNA but have limited viral transcription (no pregenomic RNA (pgRNA)), which we define as transcriptionally `silent' HBV-infected hepatocytes (tsHBiH). These cells may be functionally `cured' temporarily, but may also be the cells that reactivate. Understanding mechanisms of transcriptional silencing can elucidate intracellular processes that can be exploited to convert transcriptionally active HBV-infected hepatocytes (taHBiH) to tsHBiH and potentially lead to a functional cure. We propose comparing tsHBiH and taHBiH by dissecting single hepatocytes with single-cell laser capture microdissection (scLCM) from an established cohort of 21 HIV-HBV co-infected people who have long- standing control of HBV with DAART with archived liver tissues at two time points during DAART. In aim 1, scLCM and digital droplet PCR (ddPCR) will be used to quantify HBV replicative forms in thousands of individual hepatocytes from our cohort to determine the proportion that are tsHBiH or taHBiH. This aim further tests how HIV-1 associated immune dysregulation affects the burden of these cells. In aim 2, we test if intracellular innate immune molecules maintain tsHBiH by quantifying intracellular innate immune mRNAs. We also test alternative mechanisms such as defective virus or epigenetic silencing of cccDNA, or other viral factors. In aim 3, we use the paired liver biopsies to compare the decline rates of tsHBiH and taHBiH and test whether tsHBiH persist longer. We also determine if HIV-1 related immune dysregulation affects decline rates. Innovative aspects of this proposal include scLCM and ddPCR. Our research will provide the first quantitations of cccDNA and pgRNA in single hepatocytes in humans, and reveal mechanisms underlying HBV transcription. Relevance HBV is the leading cause of liver disease worldwide and is an important co-infection in HIV-1 infected individuals. Medicines can control chronic hepatitis B but are lifelong because they do not affect the stable genetic viral material in the principal liver cell, the hepatocyte. We aim to understand hepatocytes that are transcriptionally silently-infected (infected but not replicating virus) and the mechanisms of silencing by studying HBV viral forms from thousands of hepatocytes from HIV-HBV co-infected people. We will also determine how HIV-1 immune dysregulation affects the proportion of silently-infected hepatocytes.

慢性乙型肝炎 (CHB) 影响着全球近 4 亿人，每年约有 100 万人因慢性乙型肝炎 (CHB) 死亡肝脏疾病和肝细胞癌。 HIV-1/乙型肝炎病毒 (HBV) 双重感染很常见。 HIV-1 加速慢性乙型肝炎 (CHB) 的肝病进展，肝病是 HIV-1 死亡的主要原因接受抗逆转录病毒治疗（ART）的感染者。因此，需要治愈慢性乙型肝炎。虽然双重活跃 ART (DAART) 可控制 HBV 和 HIV-1 病毒血症，但不能治愈 CHB，因为它不能根除稳定的病毒血症来自肝细胞的 HBV 共价闭合环状 DNA (cccDNA) 形式，是复制的模板。我们的初步数据表明，一些肝细胞含有 cccDNA，但病毒转录有限（无前基因组 RNA (pgRNA))，我们将其定义为转录“沉默”的 HBV 感染肝细胞 (tsHBiH)。这些细胞可能在功能上暂时“治愈”，但也可能重新激活。理解转录沉默机制可以阐明可用于转化的细胞内过程转录活性的 HBV 感染肝细胞 (taHBiH) 转化为 tsHBiH，并可能导致功能性治愈。我们建议通过用单细胞激光捕获解剖单个肝细胞来比较 tsHBiH 和 taHBiH 对 21 名长期感染 HIV-HBV 的人进行显微解剖 (scLCM) 在 DAART 期间的两个时间点利用存档的肝组织对 HBV 进行长期控制。在目标 1 中， scLCM 和数字微滴 PCR (ddPCR) 将用于量化数千种 HBV 复制形式来自我们队列的单个肝细胞以确定 tsHBiH 或 taHBiH 的比例。这一目标进一步测试 HIV-1 相关的免疫失调如何影响这些细胞的负担。在目标 2 中，我们测试是否细胞内先天免疫分子通过量化细胞内先天免疫 mRNA 来维持 tsHBiH。我们还测试替代机制，例如缺陷病毒或 cccDNA 或其他病毒的表观遗传沉默因素。在目标 3 中，我们使用配对肝活检来比较 tsHBiH 和 taHBiH 的下降率并进行测试 tsHBiH 是否持续更长时间。我们还确定 HIV-1 相关的免疫失调是否会影响下降率。该提案的创新方面包括 scLCM 和 ddPCR。我们的研究将提供第一个定量人类单个肝细胞中的 cccDNA 和 pgRNA，并揭示 HBV 转录的机制。关联 HBV 是全世界肝病的主要原因，并且是 HIV-1 感染者的重要合并感染个人。药物可以控制慢性乙型肝炎，但药物是终生的，因为它们不会影响病情的稳定主要肝细胞（肝细胞）中的遗传病毒物质。我们的目标是了解肝细胞转录沉默感染（感染但不复制病毒）和沉默机制研究来自 HIV-HBV 共感染者的数千个肝细胞的 HBV 病毒形式。我们也会确定 HIV-1 免疫失调如何影响默默感染的肝细胞的比例。