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.

慢性肝炎B（CHB）在全球范围内影响超过2.5亿人，每年约有100万人死亡。 疾病和肝细胞癌。多达28％的艾滋病毒（PLWH）患者也患有CHB。自从 HIV增加了CHB的肝病进展，因为肝病是死亡率的主要原因 PLWH服用抗逆转录病毒疗法，开发HBV治疗是必须的。电流核（T）IDE（NUC）治疗 可以停止HBV复制，但无法治愈CHB，因为它不会消除稳定的共价关闭 循环DNA（CCCDNA），来自肝细胞的HBV复制模板。美国FDA定义HBV治疗 从血液中消除了丙型肝炎表面抗原（HBSAG）。这个定义的简单性是由 HBSAG来源的复杂性，该源来自于CCCDNA或HBV DNA的复杂性 进入宿主基因组（IDNA）。区分这两个来源对HBSAG的贡献对 目标开发治愈方法。此外，我们使用单细胞激光捕获的新技术数据 微分解（SCLCM）和液滴数字PCR（DDPCR）表明，NUC出乎意料地减少 cccDNA的PGRNA的转录，但是S的转录是否也降低了。解决 这些知识差距，我们建议确定CCCDNA和IDNA对HBSAG的贡献 在5 HIV-HBV共同感染的个体中，肝细胞水平与配对的肝组织共同感染了个体。 该提案将使用来自5个HIV-HBV共同感染的个体的存档配对肝组织，其中2个不是 3年后，在活检2时进行活检1。目标1 将在活检1的散装肝组织上使用RNA SEQ来构建表面（S）的转录图，这将使我们 确定源自CCCDNA与IDNA的S转录本的比例。后者是 之所以区分，是因为IDNA将在人类基因组中终止。然后这些地图将用于查找专业 在集成中发生的S中的断点允许开发多重DDPCR来研究单个 肝细胞。该多重DDPCR将应用于用SCLCM解剖至的100-200个单肝细胞 独特地量化来自CCCDNA与IDNA的S转录本中的每个肝细胞中的iDNA。 然后利用活检2的肝细胞了解NUC如何影响这些比例。来自AIMS 1的数据 2将与血浆定量HBSAG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先前数据相结合，或仅用于PGRNA。 我们的研究将通过使用新技术来确定HBSAG的比例来广泛影响该领域 来自CCCDNA或IDNA，这将为HBV治疗的疗法的合理设计提供信息。此外，此提案 将确定NUC是否可以在全球范围内沉默CCCDNA，或者仅适用于PGRNA。