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

• 批准号: 9974466
• 负责人: ASHWIN BALAGOPAL
• 金额: $ 79.09万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-14 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9974466
• 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; 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

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）在全球范围内影响近4亿人，每年约有100万人死亡。 肝病和肝细胞癌。 HIV-1/丙型肝炎病毒（HBV）共同感染很常见。 HIV-1 增加CHB的肝病进展，肝病是HIV-1死亡率的主要原因 接受抗逆转录病毒疗法（ART）的感染者。因此，需要治愈CHB。虽然双重活动 Art（Daart）控制HBV和HIV-1病毒血症，它无法治愈CHB，因为它不会消除稳定 HBV的共价闭合圆形DNA（CCCDNA）形式，即复制模板，来自肝细胞。我们的 初步数据表明，某些肝细胞含有CCCDNA，但病毒转录有限（无 我们将其定义为转录的“沉默” HBV感染的肝细胞（TSHBIH）。 这些细胞在功能上可以暂时“固化”，但也可能是重新激活的细胞。理解 转录沉默的机制可以阐明可以利用的细胞内过程 具有转录活性的HBV感染的肝细胞（TAHBIH），可导致功能治愈。 我们建议通过用单细胞激光捕获来比较TSHBIH和TAHBIH。 来自既定的21个HIV-HBV共同感染者的既定人群的微分解（SCLCM） DAART期间，HBV与DAART一起使用DAART和归档的肝组织。在AIM 1中， SCLCM和数字液滴PCR（DDPCR）将用于量化成千上万的HBV复制形式 来自我们同类的单个肝细胞，以确定TSHBIH或TAHBIH的比例。这个目标进一步 测试HIV-1相关的免疫失调如何影响这些细胞的负担。在AIM 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，例如有缺陷的病毒或CCCDNA的表观遗传沉默或其他病毒 因素。在AIM 3中，我们使用配对的肝活检来比较TSHBIH和TAHBIH的下降率和测试 TSHBIH是否持续更长。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。我们的研究将提供首次定量 人类单肝细胞中的CCCDNA和PGRNA的含量，并揭示了HBV转录的基础机制。 关联 HBV是全球肝病的主要原因，是HIV-1感染的重要共同感染 个人。药物可以控制慢性丙型肝炎，但终生是因为它们不会影响稳定 主要肝细胞中的遗传病毒物质，肝细胞。我们旨在了解肝细胞 转录感染的感染（感染但不复制病毒）和沉默的机制 从HIV-HBV共感染的人中研究成千上万的肝细胞的HBV病毒形式。我们也会 确定HIV-1免疫失调如何影响静静感染的肝细胞的比例。