A novel recombinant cccDNA-based mouse model with long term maintenance of rcccDNA and antigenemia
A novel recombinant cccDNA-based mouse model with long term maintenance of rcccDNA and antigenemia
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一种新型重组cccDNA小鼠模型，可长期维持rcccDNA和抗原血症
DOI：
10.1016/j.antiviral.2020.104826
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发表时间：
2020
影响因子：
7.6
通讯作者：
Zhenghong Yuan
中科院分区：
文献类型：
--
作者：
Min Wu;Cong Wang;Bisheng Shi;Zhong Fang;Boyin Qin;Xiaohui Zhou;Xiaonan Zhang;Zhenghong Yuan
乙肝病毒的共价闭合环状DNA对病毒在体内的存留起着至关重要的作用。长期以来，缺乏可靠、特异、方便的小动物模型来研究cccDNA的持久性一直是基础研究和转译研究的瓶颈。迫切需要一种能维持肝内cccDNA的小鼠模型。通过将Cre/loxP介导的重组和腺相关病毒(AAV)载体递送策略相结合，我们建立了一种新的重组CCcDNA(RcccDNA)小鼠模型。AAV-rcccDNA小鼠在转导后30周内可通过Southern blotting检测到肝内rcccDNA的长期维持。定量聚合酶链式反应可以在整个实验过程中(>；51周)检测到rcccDNA信号。此外，rcccDNA支持持续的血清抗原血症(>；72周)和肝脏内HBs Ag和HBcAg的表达(>；51周)。流式细胞仪分析和单细胞RNA测序显示，AAV-rcccDNA小鼠表现出受损的CD8+T细胞反应。同时，观察到最小的肝内炎症和纤维化。此外，三种抗乙肝病毒的化合物，转录后抑制物AKEX0007，衣壳变构调节剂Bay41-4109，以及恩替卡韦，在AAV-rcccDNA小鼠模型中被评估。这些药物的病毒标志物的变化与其作用方式一致，尽管它们都没有降低rccDNA的水平。该小鼠模型重现了免疫耐受状态，并长期维持cccDNA和抗原血症，为研究cccDNA持久性和制定最终打破耐受性和清除病毒的干预策略提供了合适的平台。
The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is critical for viral persistencein vivo. The lack of reliable, characterized and convenient small animal models for studying cccDNA persistence has long been a bottleneck for basic and translational research on HBV cure. A mouse model that can maintain intrahepatic cccDNA is urgently needed. Through combining the Cre/loxP-mediated recombination and adeno-associated virus (AAV) vector delivery strategy, we establish a novel recombinant cccDNA (rcccDNA) mouse model. AAV-rcccDNA mice supported long-term maintenance of intrahepatic rcccDNA which could be easily detected by Southern blotting within 30 weeks after transduction. Quantitative PCR could detect the rcccDNA signal throughout the experiment duration (>51 weeks). Furthermore, rcccDNA supported persistent serum antigenemia (>72 weeks) and intrahepatic HBsAg and HBcAg expression (>51 weeks). Flow cytometry analysis and single-cell RNA sequencing showed that AAV-rcccDNA mice displayed a compromised CD8+T cell response. Meanwhile, minimal intrahepatic inflammation and fibrosis were observed. Furthermore, three anti-HBV compounds, AKEX0007, a post-transcriptional inhibitor, Bay 41–4109, a capsid allosteric modulator, and Entecavir were assessed in this AAV-rcccDNA mouse model. The changes of viral markers by these drugs were consistent with their mode of action although neither of them diminished the level of rcccDNA. This mouse model recapitulated the immune tolerant state of HBV infection with long term maintenance of cccDNA and antigenemia, which will provide a suitable platform for studying cccDNA persistence and developing intervention strategies that would eventually break the tolerance and clear the virus.