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.

慢性乙型肝炎(CHB)影响着全球超过2.5亿人，每年约有100万人死于肝脏 疾病和肝细胞癌。高达28%的艾滋病毒携带者(PLWH)也患有慢性乙肝。自.以来 艾滋病毒会增加慢性乙肝的肝病进展，因为肝病是#年死亡的主要原因。 在接受抗逆转录病毒治疗的同时，开发一种治疗乙肝的药物势在必行。当前的核(T)胺(NUC)疗法 能阻止乙肝病毒复制但不能治愈慢性乙肝，因为它不能根除稳定的共价闭合 来自肝细胞的环状DNA(CcDNA)，是复制乙肝病毒的模板。美国FDA定义治疗乙肝的方法 清除血液中的乙肝表面抗原(HBs Ag)。这一定义的简单性与以下事实不符 乙肝表面抗原来源的复杂性，来源于整合的cccDNA或HBVDNA 进入宿主基因组(IDNA)。区分这两种来源对乙肝表面抗原的贡献对于 目标正在研发一种治疗方法。此外，我们的数据使用了单细胞激光捕获的新技术 显微解剖(ScLCM)和液滴数字聚合酶链式反应(DdPCR)显示核仁组成细胞意外减少 CccDNA中的pgRNA转录，但S的转录是否也被下调还不得而知。致信地址 这些认识上的差距，我们建议确定cccDNA和IDNA对单个 5例HIV-HBV混合感染者在NUC前和期间肝细胞水平的配对存档肝组织。 这项提案将使用5名艾滋病毒-乙肝病毒混合感染者的存档配对肝组织，其中2人没有 活检1时有3例在核仁组织上。活检2在~3年后获得，所有5例都在核仁组织上。目标1 将使用来自活检1的大块肝组织的rna序列来构建表面(S)转录图谱，这将使我们能够 确定来源于cccDNA和IDNA的S转录本的比例。后者是 之所以与众不同，是因为IDNA将终止于人类基因组。然后，这些地图将被用于查找主要 S的断点随着整合的发展而发生，允许开发多重ddPCR来研究单个 肝细胞。该多重ddpcr将应用于100-200个单个肝细胞的sclcm解剖。 唯一地量化来自活组织检查1的每个肝细胞的CCcDNA和IDNA中的S转录本。 然后利用活组织检查2中的肝细胞来了解核细胞如何影响这些比例。来自AIMS的数据1 2与血浆中HBs Ag定量相关。我们还将确定nucs是否会影响全球cccDNA。 通过将这些数据与我们之前从相同肝细胞获得的数据相结合，可以对pgRNA进行转录或仅对其进行转录。 我们的研究将通过使用新技术来确定乙肝表面抗原的比例，从而广泛影响该领域 来自cccDNA或IDNA，这将为乙肝治疗的合理设计提供信息。此外，这项提案 将决定nucs是否能全局沉默cccDNA，还是仅针对pgRNA。