Research Unit, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.
Research Unit, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China; Key Lab of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, China.
Antiviral Res. 2020 Aug;180:104826. doi: 10.1016/j.antiviral.2020.104826. Epub 2020 Jun 2.
The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is critical for viral persistence in 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.
乙型肝炎病毒 (HBV) 的共价闭合环状 DNA (cccDNA) 对于病毒在体内的持续存在至关重要。长期以来，缺乏可靠、特征明确且方便的用于研究 cccDNA 持续存在的小型动物模型一直是乙型肝炎病毒治愈基础和转化研究的瓶颈。迫切需要建立一种能够维持肝内 cccDNA 的小鼠模型。通过结合 Cre/loxP 介导的重组和腺相关病毒 (AAV) 载体传递策略，我们建立了一种新型重组 cccDNA (rcccDNA) 小鼠模型。AAV-rcccDNA 小鼠支持肝内 rcccDNA 的长期维持，在转导后 30 周内通过 Southern 印迹法很容易检测到，定量 PCR 可在整个实验过程中检测到 rcccDNA 信号 (>51 周)。此外，rcccDNA 支持持续的血清抗原血症 (>72 周) 和肝内 HBsAg 和 HBcAg 表达 (>51 周)。流式细胞术分析和单细胞 RNA 测序显示，AAV-rcccDNA 小鼠的 CD8 T 细胞反应受损。同时，观察到最小的肝内炎症和纤维化。此外，在这种 AAV-rcccDNA 小鼠模型中评估了三种抗乙型肝炎化合物，即 AKEX0007（一种转录后抑制剂）、Bay 41-4109（一种衣壳别构调节剂）和恩替卡韦。这些药物对病毒标志物的影响与其作用模式一致，尽管它们都没有降低 rcccDNA 的水平。该小鼠模型再现了乙型肝炎病毒感染的免疫耐受状态，具有长期维持 cccDNA 和抗原血症的能力，这将为研究 cccDNA 持续存在和开发干预策略提供合适的平台，最终打破耐受并清除病毒。
