Transcriptomic Quantitation of Hepatitis B Virus Surface Antigen from Integration

通过整合对乙型肝炎病毒表面抗原进行转录组定量

• 批准号: 10724716
• 负责人: XIAOFENG FAN
• 金额: $ 7.58万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-11 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10724716
• 关键词: Address; Antigens; Biological; Blood specimen; Characteristics; Chronic Hepatitis B; Circular DNA; Circulation; Clinical; Clinical Trials; Cohort Studies; Confidence Intervals; DNA; Data; Data Analyses; Digestion; Exclusion; Family; Genetic Transcription; Hepatitis B; Hepatitis B Infection; Hepatitis B Virus; Hepatitis B e Antigens; Hepatocarcinogenesis; Hepatology; Human; Human Genome; Immune; Immunosuppression; Incidence; Interruption; Knowledge; Legal patent; Liver; Measurement; Measures; Methods; North America; Nuclear; Outcome; Pathogenesis; Patients; Persons; Phenotype; Preparation; Primary carcinoma of the liver cells; Procedures; Proteins; Quantitative Reverse Transcriptase PCR; Research; Resistance; Sampling; Serum; Source; Specimen; Study of serum; Surface Antigens; Tissue Sample; Tissues; Transcript; Transgenic Model; Translational Research; United States National Institutes of Health; Validation; Viral; Viral Genome; Virus Integration; cohort; deoxyguanosine triphosphate; digital; experimental study; insight; member; next generation sequencing; novel; promoter; reference genome; simulation; transcriptomics; validation studies; viral DNA; viral RNA

Project Summary Hepatitis B virus (HBV) surface antigen (HBsAg) is a hallmark in patients with chronic HBV infection (CHB). Given its close association with clinical outcomes in CHB patients, HBsAg seroclearance is required in achieving an HBV functional or complete cure. Toward this direction, a major barrier is relevant to its biological sources. Besides a nuclear reservoir of covalently closed circular DNA (cccDNA), HBsAg could also be produced from cellular integration. Currently, the contribution of HBV integration to HBsAg remains elusive due to the lack of a method for quantitative measurement. Transcription from integrated HBV DNA has several characteristics, such as the retention of human sequences, an early termination, and the interruption of HBV Core antigen. These features have been used to infer the relative abundance of integration-derived HBsAg transcripts by qRT-PCR, digital droplet PCR (ddPCR), and next-generation sequencing (NGS). However, these approaches require liver tissue and have limited use with blood samples from which ~50% of HBeAg- negative patients have total HBV RNA below the lower limit of quantification of qRT-PCR. Bait-based target enrichment in NGS could enhance the sensitivity but extensive microhomology between HBV and human genomes lowers the efficiency. To address these issues, we have developed a novel method through multiple technical advances from our lab, including template-dependent multiple displacement amplification (tdMDA) (BioTechniques 2017), a novel target enrichment strategy via 7‑deaza‑dGTP‑resistant enzymatic digestion (TEED) (BMC Res Notes 2020, patented), and a read simulation to evaluate relative abundance among reference genomes (J Virol Methods 2022). The combination of tdMDA, TEED, and the read simulation, termed MATESim, was applied to five CHB patient serum samples. Integration-derived transcripts accounted for variable portions of total HBsAg transcripts, ranging from 1.8% to 94.3%. These results support the hypothesis that MATESim would be a noninvasive method to quantitate HBsAg from integration at the level of transcription. This hypothesis will be evaluated using patient specimens from the Hepatitis B Research Network (HBRN). First, we will validate MATESim by studying paired serum/liver samples. HBsAg transcripts from cccDNA and integrated HBV DNA may have different mechanisms for release to circulation. It is unknown whether their relative abundance determined by MATESim in circulation is a recapitulation, an underestimation, or an overestimation of liver data. This knowledge will help to interpret data from circulation by MATESim. Second, we will study serum samples from 59 patients with defined HBV phenotypes in the HBRN cohort. The experiment will allow us to gain insights into the magnitude and between-patient variance of integration-derived HBsAg in this patient cohort at a 95% confidence interval. Taken together, as a non- invasive method to quantitate integration-derived HBsAg, MATESim would be a turning point in translational research, clinical trials, and patient management for the globally 257 million people infected with HBV.

项目摘要 乙肝病毒表面抗原(HBs-Ag)是慢性乙肝患者的标志物。 由于它与慢性乙肝患者的临床转归密切相关，因此在以下情况下需要进行乙肝表面抗原血清清除 实现乙肝的功能性或完全治愈。朝着这个方向，一个主要的障碍是与其生物学相关的 消息来源。除了共价闭合环状DNA(CccDNA)的核心储存库外，乙肝表面抗原也可能是 从细胞整合中产生。目前，乙肝病毒整合对乙肝表面抗原的贡献仍然难以捉摸。 由于缺乏一种定量测量的方法。从整合的HBVDNA转录有几个 特征，如保留人类序列，提前终止，以及阻断乙肝病毒 核心抗原。这些特征已被用来推断整合衍生的乙肝表面抗原的相对丰度 通过qRT-PCR、数字液滴聚合酶链式反应(DdPCR)和下一代测序(NGS)获得转录本。然而， 这些方法需要肝组织，对血液样本的使用有限，从血液样本中约50%的HBeAg- 阴性患者的总HBVRNA低于qRT-PCR定量下限。诱饵目标 在NGS中的浓缩可以提高乙肝病毒和人类之间的敏感性，但具有广泛的微同源性 基因组降低了效率。为了解决这些问题，我们开发了一种新的方法，通过多次 我们实验室的技术进步，包括模板依赖的多重置换扩增(TdMDA) (BioTechniques 2017)，一种新的通过7-去氮杂-dGTP抗性酶消化的靶向浓缩策略 (Ted)(BMC RES Notes 2020，专利)，以及评估以下对象相对丰度的读取模拟 参考基因组(J Virol方法2022)。TdMDA、TED和读取模拟的组合， 对5例CHB患者血清标本进行了MATESim检测。整合衍生的成绩单被计算在内 对于总转录本中的可变部分，从1.8%到94.3%不等。这些结果支持 假设MATESim将是一种从整合水平上定量HBs Ag的非侵入性方法 抄写。这一假设将使用来自乙肝研究的患者样本进行评估。 网络(HBRN)。首先，我们将通过研究配对的血清/肝脏样本来验证MATESim。乙肝病毒表面抗原转录本 CccDNA和整合的HBVDNA可能有不同的释放到循环的机制。它是 尚不清楚由循环中的MATESim确定的它们的相对丰度是否是一种概括，以及 低估或高估肝脏数据。这一知识将有助于解释来自流通的数据 由MATESim提供。其次，我们将研究59名明确乙肝病毒表型的患者的血清样本。 HBRN队列。这项实验将使我们能够深入了解患者之间的差异和大小 整合衍生的乙肝表面抗原在该患者队列中的可信区间为95%。总而言之，作为一个非 侵入性方法定量整合衍生的乙肝表面抗原，MATESim将是翻译的一个转折点 为全球2.57亿感染乙肝病毒的人提供研究、临床试验和患者管理。