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

• 批准号: 10377407
• 负责人: ASHWIN BALAGOPAL
• 金额: $ 20.47万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-25 至 2025-03-01
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10377407
• 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 Infection; Hepatitis B Surface Antigens; Hepatitis B Therapy; 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; rational design; tool; transcriptome sequencing; viral DNA; viral RNA

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B型肝炎（CH B）影响全球超过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。以来 HIV增加了CHB的肝病进展，因为肝病是CHB患者死亡的主要原因， 艾滋病毒感染者采取抗逆转录病毒治疗，开发一种HBV治愈方法势在必行。当前的核苷（酸）治疗 可以阻止HBV复制，但不能治愈CHB，因为它不能根除稳定的共价闭合的 环状DNA（cccDNA），HBV复制的模板，来自肝细胞。美国FDA定义HBV治愈 具有消除血液中B肝炎表面抗原（HBsAg）的作用。这一定义的简单性被以下事实所掩盖： HBsAg来源的复杂性，其来源于整合的cccDNA或HBV DNA 插入宿主基因组（iDNA）。区分这两种来源对HBsAg的贡献很重要， 目标是研发解药此外，我们的数据使用单细胞激光捕获的新技术 显微切割（scLCM）和液滴数字PCR（ddPCR）表明NUC意外减少 但是S的转录是否也减少是未知的。解决 鉴于这些知识空白，我们建议在单个细胞中确定cccDNA和iDNA对HBsAg的贡献。 在NUC前和NUC期间，5名HIV-HBV合并感染者的肝细胞水平与配对存档的肝组织。 本提案将使用来自5名HIV-HBV合并感染个体（其中2名未合并）的存档配对肝组织 3例在活检1时接受NUC。在约3年后获得的活检2时，所有5个个体都在NUC上。要求1 将使用来自活检1的大块肝组织的RNA测序来构建表面（S）转录图谱，这将使我们能够 以确定源自cccDNA与iDNA的S转录物的比例。后者 因为iDNA将终止于人类基因组。这些地图将被用来寻找梅杰 S中的断点与整合一起发生，允许开发多重ddPCR来研究单一 肝细胞该多重ddPCR将应用于用scLCM解剖的100-200个单个肝细胞， 唯一地定量来自活检1的每个肝细胞中cccDNA与iDNA的S转录物。目标2将 然后利用来自活检2的肝细胞来了解NUC如何影响这些比例。数据来源：Aims 1 2与血浆HBsAg定量相关。我们还将确定NUC是否影响全局cccDNA 通过将这些数据与来自相同肝细胞的我们先前的数据相结合，来确定转录或仅针对pgRNA。 我们的研究将通过使用新技术来确定HBsAg的比例， 从cccDNA或iDNA，这将为HBV治愈疗法的合理设计提供信息。此外，该提案 将确定NUC是否可以全局沉默cccDNA或仅对pgRNA特异。