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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：
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
项目类别：
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

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）影响全球近4亿人，每年约有100万人死于慢性乙型肝炎