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 肝炎病毒（HBV）表面抗原（HBSAG）是慢性HBV感染（CHB）患者的标志。 鉴于其与CHB患者的临床结局的密切关联，需要HBSAG血清清除率 实现HBV功能或完整治疗。朝这个方向朝着这个方向，主要障碍与其生物学有关 来源。除了共价闭合圆形DNA（CCCDNA）的核储存库外，HBSAG也可以是 由细胞整合产生。目前，HBV集成对HBSAG的贡献仍然难以捉摸 由于缺乏定量测量方法。来自集成HBV DNA的转录具有多个 特征，例如人类序列的保留，早期终止和HBV的中断 核心抗原。这些功能已用于推断集成衍生的HBSAG的相对抽象 QRT-PCR，数字液滴PCR（DDPCR）和下一代测序（NGS）的笔录。然而， 这些方法需要肝组织，并且血液样本的使用有限，其中约50％的hbeag- 阴性患者的总HBV RNA低于QRT-PCR定量的下限。基于诱饵的目标 NGS的富集可以增强HBV与人之间的敏感性但广泛的微学学 基因组降低了效率。为了解决这些问题，我们通过多个开发了一种新颖的方法 我们实验室的技术进步，包括依赖模板的多重位移扩增（TDMDA） （Biotechniques 2017），一种新型的目标富集策略，该策略通过7 − deaza − dgtp-抗性酶消化 （TEED）（BMC Res Notes 2020，获得专利）和读取模拟，以评估相对丰度 参考基因组（J Virol方法2022）。 TDMDA，TEED和读取模拟的组合， 称为Matesim，应用于五个CHB患者血清样品。集成衍生的成绩单被解释了 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 假设Matesim将是一种无创的方法，可以从在 转录。该假设将使用丙型肝炎研究的患者标本进行评估 网络（HBRN）。首先，我们将通过研究配对的血清/肝样品来验证Matesim。 HBSAG转录本 来自CCCDNA和集成的HBV DNA可能具有不同的释放机制。这是 尚不清楚其相对丰度是否由循环中的Matesim确定是一种概括， 低估或高估肝脏数据。这些知识将有助于解释流通的数据 由Matesim。其次，我们将研究来自59例HBV表型患者的血清样品 HBRN队列。该实验将使我们能够深入了解大小和患者之间的方差 该患者队列中的集成衍生的HBSAG的置信区间为95％。一起，作为非 - 量化集成衍生的HBSAG的侵入性方法，Matesim将是翻译的转折点 感染HBV的全球2.57亿人的研究，临床试验和患者管理。