Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, New Mexico, USA.
JCI Insight. 2020 Oct 2;5(19):140584. doi: 10.1172/jci.insight.140584.
There is no cure for the more than 270 million people chronically infected with HBV. Nucleos(t)ide analogs (NUCs), the mainstay of anti-HBV treatment, block HBV reverse transcription. NUCs do not eliminate the intranuclear covalently closed circular DNA (cccDNA), from which viral RNAs, including pregenomic RNA (pgRNA), are transcribed. A key gap in designing a cure is understanding how NUCs affect HBV replication and transcription because serum markers yield an incomplete view of intrahepatic HBV. We applied single-cell laser capture microdissection and droplet digital PCR to paired liver biopsies collected from 5 HBV/HIV-coinfected persons who took NUCs over 2-4 years. From biopsy 1 to 2, proportions of HBV-infected hepatocytes declined with adherence to NUC treatment (P < 0.05); we extrapolated that eradication of HBV will take over 10 decades with NUCs in these participants. In individual hepatocytes, pgRNA levels diminished 28- to 73-fold during NUC treatment, corresponding with decreased tissue HBV core antigen staining (P < 0.01). In 4 out of 5 participants, hepatocytes with cccDNA but undetectable pgRNA (transcriptionally inactive) were present, and these were enriched in 3 participants during NUC treatment. Further work to unravel mechanisms of cccDNA transcriptional inactivation may lead to therapies that can achieve this in all hepatocytes, resulting in a functional cure.
有超过 2.7 亿人慢性感染 HBV，目前尚无治愈方法。核苷（酸）类似物（NUC）是抗 HBV 治疗的主要手段，可阻断 HBV 逆转录。NUC 并不能消除核内共价闭合环状 DNA（cccDNA），HBV 的 RNA，包括前基因组 RNA（pgRNA），就是从这里转录的。设计治愈方法的一个关键空白是了解 NUC 如何影响 HBV 的复制和转录，因为血清标志物对肝内 HBV 的观察并不完整。我们应用单细胞激光捕获显微切割和液滴数字 PCR 技术，对 5 名接受 NUC 治疗超过 2-4 年的 HBV/HIV 合并感染者的配对肝活检组织进行了研究。从活检 1 到活检 2，随着 NUC 治疗的依从性提高，HBV 感染的肝细胞比例下降（P < 0.05）；我们推断，在这些参与者中，用 NUC 治疗需要超过 10 个十年才能根除 HBV。在单个肝细胞中，pgRNA 水平在 NUC 治疗期间下降了 28 至 73 倍，与组织 HBV 核心抗原染色减少（P < 0.01）相对应。在 5 名参与者中的 4 名中，存在含有 cccDNA 但 pgRNA 不可检测（转录失活）的肝细胞，在 3 名参与者中，在 NUC 治疗期间，这些肝细胞更丰富。进一步研究 cccDNA 转录失活的机制，可能会开发出能使所有肝细胞达到这一效果的治疗方法，从而实现功能性治愈。
