Identifying the source of hepatitis B surface antigen in people with hepatitis B-HIV co-infection

鉴定乙型肝炎-HIV 合并感染者的乙型肝炎表面抗原来源

• 批准号: 10448435
• 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: 2021-07-12 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10448435
• 关键词: Address; Affect; Archives; Biological Assay; Biopsy; Blood; C-terminal; CD4 Positive T Lymphocytes; Cell Count; Cessation of life; Chronic; Chronic Active Hepatitis; Chronic Hepatitis B; Circular DNA; Cirrhosis; Complementary DNA; DNA; Data; Detection; Development; Disease; Disease Progression; Foundations; Future; Genetic Transcription; Genome; Genomics; HIV; HIV-1; Hepatitis B; Hepatitis B Infection; Hepatitis B Surface Antigens; Hepatitis B Therapy; Hepatitis B Virus; Hepatitis B e Antigens; Hepatocyte; Human Genome; Immune; In Vitro; Individual; Knowledge; Length; Liver; Liver diseases; Malignant neoplasm of liver; Maps; Messenger RNA; Open Reading Frames; Outcome; Persons; Plasma; Poly(A) Tail; Primary carcinoma of the liver cells; Production; Research; Risk; Role; Sampling; Serum; Source; Surface; T-Cell Depletion; Techniques; Time; Tissues; Transcript; Translating; United States National Institutes of Health; Vertebral column; Viral; analog; antiretroviral therapy; base; co-infection; design; detection limit; digital; env Gene Products; innovation; liver biopsy; mortality; novel; pgRNA; rational design; therapeutic target; tool; transcriptome sequencing

Project Summary 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 control 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. In addition, the US FDA defines HBV cure has elimination of total hepatitis B surface antigen (tHBsAg) from blood. The simplicity of this definition is belied by the complexity of the source of tHBsAg, 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 droplet digital PCR (ddPCR) demonstrate that NUCs unexpectedly decrease transcription of pgRNA from cccDNA, but whether transcription of S mRNAs, the transcripts that encode for tHBsAg, is also reduced is unknown. To address these knowledge gaps, we propose to determine the contribution of cccDNA and iDNA to tHBsAg from 69 PLWH with different stages of CHB of whom 60 have paired biopsies. In a subset of these individuals, we will examine single hepatocytes to determine the proportions of hepatocytes with iDNA and cccDNA. The 69 individuals (129 biopsies since 60 have paired biopsies) in this proposal having varying stages of HBV infection including immune active CHB (HBeAg+ and HBeAg neg), inactive CHB, and occult hepatitis B. Aim 1 will use RNA seq on bulk liver tissue from 6 individuals with CHB to construct surface (S) mRNA maps, which will allow us to determine the proportion of S mRNA that originate from cccDNA versus iDNA. The latter are distinguished because iDNA will terminate in the human genome, truncating the viral sequence at its 3’ end. The maps will then be used to find major breakpoints in S mRNAs that occur with integration, allowing development of a multiplex ddPCR to study 69 bulk liver tissues and single hepatocytes from a subset of individuals. Aim 2 will interrogate liver biopsies from the 60 individuals with longitudinal biopsies during which time they were on NUCs to understand how NUCs affect these proportions. Data from Aims 1 and 2 will be correlated with plasma quantitative HBsAg and with circulating amounts of Large, Medium, and Small HBsAg. We will also determine if CD4+ T cell depletion affects the proportion of HBsAg that derives from iDNA. Our research will broadly impact the field by using novel techniques to determine the proportions of tHBsAg from iDNA or cccDNA, which will inform the rational design of therapies for HBV cure.

项目摘要 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 可以控制HBV复制，但无法治愈CHB，因为它不会放射线稳定的稳定关闭 圆形DNA（CCDNA），来自肝细胞的HBV复制模板。此外，美国FDA定义了 HBV治疗从血液中消除了总丙型肝炎表面抗原（THBSAG）。简单性 定义是由THBSAG来源的复杂性所掩盖的，THBSAG的来源源自CCCDNA或HBV 集成到宿主基因组（IDNA）中的DNA。区分这两个来源对 HBSAG对于靶向开发治疗很重要。此外，我们使用液滴数字的新技术数据 PCR（DDPCR）表明，NUC会意外地减少CCCDNA的PGRNA的转录，但 S mRNA的转录（编码THBSAG的转录物）是否也降低。到 解决这些知识差距，我们建议确定CCCDNA和IDNA对THBSAG的贡献 69个PLWH具有不同的CHB阶段的PLWH，其中60个具有成对的活检。在这些人的子集中，我们 将检查单个肝细胞，以确定肝细胞与IDNA和CCCDNA的比例。 在此提案中，有69个人（自60个以来有129个活检具有配对活检），其阶段有不同的HBV 感染包括免疫反应性CHB（HBEAG+和HBEAG NEG），无活性CHB和隐匿性乙型肝炎。AIM 1 将在6个患有CHB个体的散装肝组织上使用RNA SEQ来构建表面mRNA图，其中 将使我们能够确定源自CCCDNA与IDNA的S mRNA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，从而在其3'末端截断病毒序列。 然后，这些地图将用于在集成中发生的S mRNA中找到主要的断点，从而允许 从一部分的子集中开发多重DDPCR以研究69个散装肝组织和单肝细胞 个人。 AIM 2将从60名纵向活检的人那里询问肝活检 他们在NUC上了解NUC如何影响这些比例。 AIM 1和2的数据将是 与血浆定量HBSAG以及循环量的大，中和小HBSAG相关。 我们还将确定CD4+ T细胞部署是否影响来自IDNA的HBSAG的比例。 我们的研究将通过使用新技术来确定THBSAG的比例，从而广泛影响该领域 来自IDNA或CCCDNA，这将为HBV治疗的疗法的合理设计提供信息。