A novel recombinant cccDNA-based mouse model with long term maintenance of rcccDNA and antigenemia

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.