Transcriptomic Quantitation of Hepatitis B Virus Surface Antigen from Integration

项目摘要 B型肝炎病毒（HBV）表面抗原（HBsAg）是慢性HBV感染（CH B）患者的标志。 鉴于其与CHB患者的临床结局密切相关， 实现HBV功能性或完全治愈。朝着这个方向，一个主要的障碍与其生物学特性有关。 源除了共价闭合环状DNA（cccDNA）的核储库外，HBsAg还可以是 由细胞整合而成。目前，HBV整合对HBsAg的贡献仍然是难以捉摸的 由于缺乏定量测量的方法。从整合的HBV DNA转录有几个 特征，如保留人类序列，提前终止和HBV的中断， 核心抗原。这些特征已被用于推断整合来源的HBsAg的相对丰度 通过qRT-PCR、数字液滴PCR（ddPCR）和下一代测序（NGS）检测转录物。然而，在这方面， 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- 阴性患者的总HBV RNA低于qRT-PCR的定量下限。诱饵靶 富集NGS可提高敏感性，但HBV与人之间存在广泛的微同源性 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， 我们实验室的技术进步，包括模板依赖性多重置换扩增（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，专利），以及读取模拟，以评估 参考基因组（J Virol Methods 2022）。tdMDA、TEED和读取模拟的组合， 称为MATESim的方法应用于5个CHB患者血清样品。整合衍生的转录本占 对于总HBsAg转录物的可变部分，范围为1.8%至94.3%。这些结果支持这一 假设MATESim是一种非侵入性方法，可在整合水平上定量HBsAg， 转录。将使用来自B型肝炎研究的患者标本评价该假设 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。HBsAg转录本 从cccDNA和整合的HBV DNA释放到循环中可能具有不同的机制。是 不知道它们的相对丰度是否由MATESim在流通中确定是一个重演， 低估或高估肝脏数据。这些知识将有助于解释流通数据 的MATESim。第二，我们将研究59例具有明确HBV表型的患者的血清样本， HBRN队列。该实验将使我们能够深入了解患者之间的差异 在95%置信区间下，该患者队列中整合来源的HBsAg。作为一个非- 定量整合来源HBsAg的侵入性方法，MATESim将是转化的转折点 为全球2.57亿HBV感染者提供研究、临床试验和患者管理。

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.