Quantifying the frequency and diversity of spliced HBV mRNAs in HIV-HBV co-infection and their role in modulating viral transcription and host immune responses

量化 HIV-HBV 合并感染中 HBV mRNA 剪接的频率和多样性及其在调节病毒转录和宿主免疫反应中的作用

• 批准号: 10761937
• 负责人: TANNER GRUDDA
• 金额: $ 4.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10761937
• 关键词: Acceleration; Address; Affect; Ancillary Study; Antiviral Therapy; Archives; Biological Assay; Biological Markers; Blood; Cause of Death; Cell Line; Cell Nucleus; Cells; Chronic; Chronic Hepatitis B; Circular DNA; Code; Cohort Studies; Development; Disease; Disease Progression; Excision; Frequencies; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Genomics; Goals; HIV; HepG2; Hepatitis; Hepatitis B; Hepatitis B Infection; Hepatitis B Surface Antigens; Hepatitis B Virus; Hepatocyte; Human; Immune response; In Vitro; Individual; Interferons; Interruption; Lead; Link; Liver; Liver diseases; Measures; Messenger RNA; Metabolic Clearance Rate; Methods; Modeling; Molecular; Mus; N-terminal; Participant; Pathway interactions; Pegylated Interferon Alfa; Persons; Polymerase; Population; Primary carcinoma of the liver cells; Protein Splicing; Proteins; RNA; RNA Interference; RNA Splicing; Reporting; Research; Resistance; Reverse Transcription; Risk; Role; Sampling; Serum; Signal Transduction; Small Interfering RNA; Surface Antigens; Techniques; Tissues; Transcript; Up-Regulation; Validation; Viral; Viral Proteins; Viral hepatitis; Viremia; Virus Diseases; Virus Replication; alternative treatment; analog; antagonist; co-infection; comparative; deep sequencing; differential expression; digital; drug development; knock-down; liver injury; mortality; new therapeutic target; novel; overexpression; response; side effect; therapy resistant; tool; viral DNA; viral RNA

Over 316 million people are living with chronic hepatitis B and as many as one quarter of people living with HIV have hepatitis B virus (HBV) co-infection. HIV-HBV co-infection increases levels of HBV replication, liver disease development, and the risk of hepatocellular carcinoma. The key to HBV persistence is nucleus- resident, long-lived covalently closed circular DNA (cccDNA) coding for all viral proteins. Nucleos(t)ide analogue therapy (NUC) effectively reduces HBV DNA in the serum by halting reverse transcription of pregenomic RNA (pgRNA). Recently, NUC therapy has been associated with cccDNA transcriptional silencing leading to reduced pgRNA levels in the liver and HBV RNA in the serum, however cccDNA quantities across the liver remain stable. Efforts to develop a cure for HBV focus on the removal or long term control of cccDNA activity, the latter yielding a functional cure. An alternative treatment to NUC is pegylated interferon-α (PEG- IFN), a potent antiviral therapy with comparatively greater rates of functional cure (7%), defined as a durable loss of serum HBV surface antigen (HBsAg). A study following participants before and after PEG-IFN found that treatment non-responders had elevated proportions of spliced HBV (spHBV) DNA from total HBV DNA, supporting a role of spHBV in modulating IFN responsiveness. There are 20 identified spHBV transcripts derived from pgRNA, a subset of which encode noncanonical HBV proteins. A limited number of studies have demonstrated that spHBV expression disrupts IFN response signaling and possibly alters cccDNA transcription. Given their putative role in modulating the host immune response and viral transcription, we propose to compare spHBV in HBV mono-infection and HIV-HBV co-infection. Combining use of a novel multiprobe multiplex droplet digital PCR with direct sequencing, we will characterize spHBV in HIV-HBV co- infection and HBV monoinfection in serum from individuals in the MACS/WIHS Combined Cohort Study. We expect an enrichment of spHBV RNA within total HBV RNA in co-infection compared to mono-infection. Additionally, we will model the decay of spHBV during NUC in HIV-HBV co-infection and compare spHBV expression in the same person’s liver and blood. Additionally, we will perform deep sequencing of hepatocytes with high vs low HBV transcription in an HIV-HBV coinfection ancillary study of the Hepatitis B Research Network, focusing on innate response pathway genes. We will then overexpress candidate spHBV that are likely to affect IFN responses in HepG2-NTCP cells. Conversely, we will use siRNA to knockdown candidate spHBV and measure IFN responses in an HBV infected HepG2-NTCP cells. Using this model in the context of HBV infection, we will measure the interplay between cccDNA transcription, spHBV expression, and innate signaling. This study will be the first characterization of spHBV in HIV-HBV co-infection and has the goal of identifying novel therapeutic targets specifically affecting cccDNA transcription in CHB.

超过 3.16 亿人患有慢性乙型肝炎，多达四分之一的人患有艾滋病毒 乙型肝炎病毒 (HBV) 合并感染 HIV-HBV 合并感染会增加 HBV 复制水平，肝脏。 疾病发展和肝细胞癌的风险 HBV 持续存在的关键是细胞核。 编码所有病毒蛋白的长寿命共价闭合环状 DNA (cccDNA)。 类似疗法 (NUC) 通过阻止 HBV DNA 的逆转录，有效减少血清中的 HBV DNA 最近，NUC 疗法与 cccDNA 转录沉默相关。 导致肝脏中的 pgRNA 水平和血清中的 HBV RNA 水平降低，但是 cccDNA 数量 开发治疗 HBV 的方法的重点是消除或长期控制 cccDNA。 NUC 的另一种治疗方法是聚乙二醇化干扰素-α（PEG-）。 IFN），一种有效的抗病毒疗法，具有相对较高的功能性治愈率（7％），被定义为持久的 一项针对 PEG-IFN 治疗前后参与者的研究发现，血清 HBV 表面抗原 (HBsAg) 丢失。 治疗无反应者的 HBV DNA 总量中剪接的 HBV (spHBV) DNA 比例升高， 支持 spHBV 在调节 IFN 反应性中的作用 有 20 个已识别的 spHBV 转录本。 衍生自 pgRNA，其中的一个子集编码非典型 HBV 蛋白。 spHBV 表达会破坏 IFN 反应信号并可能改变 cccDNA 鉴于它们在调节宿主免疫反应和病毒转录中的假定作用，我们 提议比较 spHBV 在 HBV 单一感染和 HIV-HBV 合并感染中的新型联合使用。 多探针多重液滴数字 PCR 与直接测序，我们将表征 HIV-HBV 共存中的 spHBV MACS/WIHS 联合队列研究中个体血清中的感染和 HBV 单一感染。 与单一感染相比，预计合并感染时总 HBV RNA 中 spHBV RNA 会富集。 此外，我们将模拟 HIV-HBV 共感染 NUC 期间 spHBV 的衰减，并比较 spHBV 此外，我们将对肝细胞进行深度测序。 乙型肝炎研究的 HIV-HBV 共感染辅助研究中 HBV 转录的高与低 网络，重点关注先天反应途径基因，然后我们将过度表达候选 spHBV。 可能会影响 HepG2-NTCP 细胞中的 IFN 反应，我们将使用 siRNA 来敲除候选者。 spHBV 并测量 HBV 感染的 HepG2-NTCP 细胞中的 IFN 反应。 HBV 感染，我们将测量 cccDNA 转录、spHBV 表达和先天性之间的相互作用 这项研究将是 HIV-HBV 共感染中 spHBV 的首次表征，其目标是 确定专门影响 CHB 中 cccDNA 转录的新治疗靶点。