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

基本信息

批准号：
10326630
负责人：
ASHWIN BALAGOPAL
金额：
$ 24.56万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-12 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10326630
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 Surface Antigens Hepatitis B Therapy Hepatitis B Virus Hepatitis B e Antigens Hepatocyte Human Genome Immune In Vitro Individual Infection 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 therapeutic target therapy design tool transcriptome sequencing

项目摘要

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.

项目概要慢性乙型肝炎 (CHB) 影响着全球超过 2.5 亿人，每年约有 100 万人死于肝脏疾病高达 28% 的艾滋病毒感染者 (PLWH) 也患有慢性乙型肝炎。 HIV 会加速慢性乙型肝炎 (CHB) 的肝病进展，因为肝病是导致慢性乙型肝炎 (CHB) 死亡的主要原因正在接受抗逆转录病毒治疗的感染者，开发治疗乙肝病毒的药物势在必行。可以控制 HBV 复制，但不能治愈 CHB，因为它不能消除稳定的共价闭合此外，美国 FDA 还定义了来自肝细胞的环状 DNA (cccDNA)，它是 HBV 复制的模板。乙型肝炎治愈方法是消除血液中的总乙型肝炎表面抗原 (tHBsAg)。 tHBsAg 来源的复杂性相信这一定义，它源自 cccDNA 或 HBV 整合到宿主基因组中的 DNA (iDNA) 区分这两种来源的贡献。此外，我们使用液滴数字新技术获得的数据对于靶向治疗非常重要。 PCR (ddPCR) 证明 NUC 出乎意料地减少了 cccDNA 中 pgRNA 的转录，但是 S mRNA（编码 tHBsAg 的转录本）的转录是否也减少尚不清楚。为了解决这些知识空白，我们建议确定 cccDNA 和 iDNA 对 tHBsAg 的贡献我们对 69 名患有不同阶段 CHB 的 PLWH 进行了配对活检。将检查单个肝细胞以确定具有 iDNA 和 cccDNA 的肝细胞的比例。该提案中的 69 个人（自 60 人以来已进行了配对活检的 129 人）患有不同阶段的 HBV 感染，包括免疫活动性 CHB（HBeAg+ 和 HBeAg 阴性）、非活动性 CHB 和隐匿性乙型肝炎。目标 1 将使用 RNA seq 对 6 名 CHB 患者的大块肝组织构建表面 (S) mRNA 图谱，将使我们能够确定源自 cccDNA 与 iDNA 的 S mRNA 的比例。之所以区分，是因为 iDNA 将在人类基因组中终止，从而在 3' 端截断病毒序列。然后，这些图谱将用于查找 S mRNA 中发生整合的主要断点，从而允许开发多重 ddPCR 来研究 69 个大块肝组织和来自一个子集的单个肝细胞目标 2 将询问 60 名个体的肝脏活检，在此期间进行纵向活检。他们在 NUC 上了解 NUC 如何影响目标 1 和 2 的数据。与血浆 HBsAg 定量以及大、中、小 HBsAg 的循环量相关。我们还将确定 CD4+ T 细胞耗竭是否会影响源自 iDNA 的 HBsAg 比例。我们的研究将通过使用新技术来确定 tHBsAg 的比例来广泛影响该领域来自 iDNA 或 cccDNA，这将为乙肝治疗的合理设计提供信息。