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.

项目概要 乙型肝炎病毒 (HBV) 表面抗原 (HBsAg) 是慢性 HBV 感染 (CHB) 患者的标志。 鉴于 HBsAg 血清清除与 CHB 患者的临床结果密切相关， 实现乙型肝炎功能性或完全治愈，一个主要障碍与其生物学有关。 除了共价闭合环状 DNA (cccDNA) 的核库外，HBsAg 也可能是来源。 目前，HBV 整合对 HBsAg 的贡献仍然难以捉摸。 由于缺乏定量测量整合的 HBV DNA 转录的方法，有几种方法。 特征，例如保留人类序列、提前终止和中断 HBV 这些特征已被用来推断整合衍生的 HBsAg 的相对丰度。 然而，通过 qRT-PCR、数字微滴 PCR (ddPCR) 和下一代测序 (NGS) 来检测转录本。 这些方法需要肝组织，并且对血液样本的使用有限，其中约 50% 的 HBeAg- 阴性患者的 HBV RNA 总量低于基于 qRT-PCR 的目标定量下限。 NGS 富集可以提高敏感性，但 HBV 和人类之间存在广泛的微同源性 为了解决这些问题，我们通过多种方法开发了一种新方法。 我们实验室的技术进步，包括模板依赖性多重位移扩增 (tdMDA) (BioTechniques 2017)，一种通过 7-deaza-dGTP 抗性酶消化的新型靶标富集策略 (TEED)（BMC Res Notes 2020，已获得专利），以及用于评估相对丰度的读取模拟 参考基因组（J VirolMethods 2022）tdMDA、TEED 和读取模拟的组合， 称为 MATESim，应用于 5 个 CHB 患者血清样本的整合衍生转录本。 对于总 HBsAg 转录本的可变部分，范围从 1.8% 到 94.3% 这些结果支持了这一点。 假设 MATESim 将是一种从整合水平定量 HBsAg 的无创方法 该假设将使用乙型肝炎研究的患者标本进行评估。 首先，我们将通过研究配对的血清/肝脏样本来验证 MATESim。 cccDNA 和整合的 HBV DNA 可能具有不同的释放到循环的机制。 不知道流通中的 MATESim 确定的它们的相对丰度是否是重演、 低估或高估肝脏数据将有助于解释循环数据。 其次，我们将研究 59 名具有明确 HBV 表型的患者的血清样本。 该实验将使我们能够深入了解患者之间的差异程度和差异。 该患者队列中整合衍生的 HBsAg 的置信区间为 95%，作为非-。 MATESim 是量化整合衍生的 HBsAg 的侵入性方法，将成为转化的转折点 为全球 2.57 亿 HBV 感染者提供研究、临床试验和患者管理。