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B型肝炎，多达四分之一的艾滋病毒感染者 患有B型肝炎病毒（HBV）合并感染。HIV-HBV合并感染增加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- 常驻的、长寿命的共价闭合环状DNA（cccDNA），编码所有病毒蛋白。核苷 类似物治疗（NUC）通过阻断HBV DNA的逆转录， 前基因组RNA（pgRNA）。最近，NUC治疗与cccDNA转录沉默有关 导致肝脏中的pgRNA水平和血清中的HBV RNA水平降低，然而， 肝脏保持稳定。开发HBV治愈的努力集中在cccDNA的去除或长期控制上 活性，后者产生功能治愈。NUC的替代治疗是聚乙二醇化干扰素-α（PEG-α）。 干扰素），一种有效的抗病毒治疗，具有相对较高的功能治愈率（7%），被定义为持久的 血清HBV表面抗原（HBsAg）丢失。一项对PEG-IFN前后参与者的研究发现， 治疗无应答者中剪接的HBV（spHBV）DNA占总HBV DNA的比例升高， 支持spHBV在调节IFN应答中的作用。有20个识别的spHBV转录本 来源于pgRNA，其子集编码非典型HBV蛋白。有限数量的研究 表明spHBV表达破坏IFN应答信号传导，并可能改变cccDNA 转录。考虑到它们在调节宿主免疫应答和病毒转录中的假定作用， 建议比较HBV单一感染和HIV-HBV合并感染中的spHBV。结合使用一本小说 多探针多重液滴数字PCR与直接测序，我们将在HIV-HBV共感染中表征spHBV， MACS/WIHS联合队列研究中个体血清中HBV感染和HBV单一感染的研究。我们 预期与单一感染相比，合并感染中总HBV RNA中spHBV RNA的富集。 此外，我们将模拟HIV-HBV合并感染NUC期间spHBV的衰减， 在同一个人的肝脏和血液中表达。此外，我们将对肝细胞进行深度测序， 在B型肝炎研究的一项HIV-HBV合并感染辅助研究中， 网络，专注于先天反应途径基因。然后我们将过表达候选的spHBV， 可能影响HepG 2-NTCP细胞中的IFN应答。相反，我们将使用siRNA敲低候选基因， spHBV和测量HBV感染的HepG 2-NTCP细胞中的IFN应答。在以下背景下使用此模型 HBV感染后，我们将测量cccDNA转录、spHBV表达和先天性HBV感染之间的相互作用。 信号这项研究将是HIV-HBV合并感染中spHBV的首次表征，其目标是 鉴定特异性影响CHB中cccDNA转录的新治疗靶标。