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: 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
• 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

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)影响全球近4亿人，每年约有100万人死于 肝病和肝细胞癌。HIV-1/乙肝病毒(乙肝)混合感染很常见。HIV-1 增加慢性乙型肝炎的肝病进展，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，需要一种治疗慢性乙肝的方法。虽然是双重活跃的 抗逆转录病毒治疗(DAART)控制了乙肝病毒和HIV-1病毒血症，但不能治愈慢性乙肝，因为它不能根除稳定的 来自肝细胞的共价闭合环状DNA(CccDNA)形式的乙肝病毒，用于复制的模板。我们的 初步数据显示，一些肝细胞含有cccDNA，但病毒转录有限(NO 前基因组RNA(PgRNA)，我们将其定义为转录上“沉默的”乙肝病毒感染的肝细胞(TsHBiH)。 这些细胞可能在功能上暂时“治愈”，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细胞内的过程，这些过程可以被利用来转化 转录活性的乙肝病毒感染的肝细胞(TaHBiH)与tsHBiH结合，有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 显微解剖(ScLCM)来自21名HIV-HBV共感染者，这些人长期- 在DAART过程中的两个时间点，用存档的肝组织作为DAART中乙肝病毒的常备对照。在目标1中， ScLCM和数字滴状聚合酶链式反应(DdPCR)将被用来量化数以千计 我们队列中的单个肝细胞来确定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 细胞内天然免疫分子通过定量细胞内天然免疫mRNAs来维持tsHBiH。我们 也要测试替代机制，如缺陷病毒或cccDNA的表观遗传沉默，或其他病毒 各种因素。在目标3中，我们使用配对的肝活检来比较tsHBiH和taHBiH的下降率，并测试 THBiH是否持续更久。我们还确定了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批量化数据 人单个肝细胞中cccDNA和pgRNA的表达，并揭示了乙肝病毒转录的机制。 相关性 乙肝病毒是世界范围内引起肝病的主要原因，也是HIV-1感染者的重要混合感染。 个人。药物可以控制慢性乙肝，但因为它们不影响稳定，所以是终生的 主要肝细胞中的遗传性病毒物质，即肝细胞。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)和沉默的机制 从HIV-HBV混合感染者的数千个肝细胞中研究HBV病毒的形式。我们还将 确定HIV-1免疫失调如何影响静默感染的肝细胞比例。