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3.6亿人患有慢性乙型肝炎，多达四分之一的人患有艾滋病毒 患有丙型肝炎病毒（HBV）共感染。 HIV-HBV共感染增加了HBV复制水平，肝脏 疾病的发展以及肝细胞癌的风险。 HBV持久性的关键是核 - 驻留的，长寿的共价闭合圆形DNA（CCCDNA）编码所有病毒蛋白。核（T）IDE 模拟疗法（NUC）通过停止逆转录，有效地降低了血清中HBV DNA 基因组RNA（PGRNA）。最近，NUC治疗与CCCDNA转录沉默有关 导致血清中肝脏和HBV RNA的PGRNA水平降低，但是CCCDNA的数量遍布 肝脏保持稳定。努力开发HBV的努力专注于CCCDNA的去除或长期控制 活性，后者产生功能性治疗。 NUC的另一种治疗方法是pegypated干扰素-α（PEG- IFN），具有功能疗法速率较高的潜在抗病毒疗法（7％），被定义为耐用 血清HBV表面抗原（HBSAG）的丧失。在发现PEG-IFN之前和之后的参与者的一项研究 该治疗无反应器的比例升高，来自总HBV DNA的剪接HBV（SPHBV）DNA， 支持SPHBV在调节IFN响应能力中的作用。有20个已识别的SPHBV转录本 源自PGRNA，该子集编码非规范HBV蛋白。有限的研究有 证明SPHBV表达会破坏IFN响应信号，并可能改变CCCDNA 转录。鉴于它们在调节宿主免疫反应和病毒转录中的推定作用，我们 比较HBV单感染和HIV-HBV共同感染中SPHBV的建议。结合小说的使用 多功能多重液滴数字PCR具有直接测序，我们将表征HIV-HBV共同的SPHBV MAC/WIHS组合队列研究中的个体的血清中感染和HBV单感染。我们 与单感染相比，共同感染中总HBV RNA内的SPHBV RNA富集。 此外，我们将在HIV-HBV共感染中对SPHBV的衰减进行建模，并比较SPHBV 在同一人的肝脏和血液中表达。此外，我们将对肝细胞进行深层测序 在HIV-HBV共感染辅助研究中，具有高HBV转录的高HBV转录。 网络，专注于先天响应途径基因。然后，我们将过表达候选SPHBV 可能影响HEPG2-NTCP细胞中的IFN响应。相反，我们将使用siRNA击倒候选人 SPHBV并测量感染HEPG2-NTCP细胞的HBV中的IFN反应。在上下文中使用此模型 HBV感染，我们将测量CCCDNA转录，SPHBV表达和先天的相互作用 信号。这项研究将是SPHBV在HIV-HBV共感染中的第一个特征，其目标是 识别特异性影响CCCDNA转录的新型热目标。