AAV-HBV mouse model shows similar exhaustion marker pattern to chronic HBV patients at single-cell level.
Background and Aims: Unresolved hepatitis B virus (HBV) infection leads to a progressive state of immune exhaustion that impairs resolution of infection, leading to chronic infection (CHB). The immune-competent AAV-HBV mouse is a common HBV preclinical immune competent model, though a comprehensive characterization of the liver immune microenvironment and its translatability to human infection is still lacking. We investigated the intrahepatic immune profile of the AAV-HBV mouse model at a single-cell level and compared with data from CHB patients in immune tolerant (IT) and immune active (IA) clinical stages.  Methods: Immune exhaustion was profiled through an iterative subclustering approach for cell-typing analyses of single-cell RNA-sequencing data in CHB donors and compared to the AAV-HBV mouse model 24-weeks post-transduction to assess its translatability. This was validated using an exhaustion flow cytometry panel at 40 weeks post-transduction. Results: Using single-cell RNA-sequencing, CD8 pre-exhausted T-cells with self-renewing capacity (TCF7+), and terminally exhausted CD8 T-cells (TCF7-) were detected in the AAV-HBV model. These terminally exhausted CD8 T-cells (expressing Pdcd1, Tox, Lag3, Tigit) were significantly enriched versus control mice and independently identified through flow cytometry. Importantly, comparison to CHB human data showed a similar exhausted CD8 T-cell population in IT and IA donors, but not in uninfected individuals.  Conclusions: Long term high titer AAV-HBV mouse liver transduction led to T-cell exhaustion, as evidenced by expression of classical immune checkpoint markers at mRNA and protein levels. In both IT and IA donors, a similar CD8 exhausted T-cell population was identified, with increased frequency observed in IA donors. These data support the use of the AAV-HBV mouse model to study T-cell exhaustion in HBV infection and the effect of immune-based therapeutic interventions. Overall design: Male C57BL/6 mice (4–5-week-old, Wuxi Apptec, Shanghai, China) were either transduced via the tail vein with 1x1011 viral genome equivalents (vge) per mouse of rAAV-HBV1.3 (n=5)  (FivePlus Molecular Medicine Institute, Beijing, China) or rAAV vector control (n=5), which contained an empty rAAV capsid and rAAV containing a short sequence of the CMV promoter and BGH poly A tail flanked with inverted AAV-ITR or kept naïve (n=5). IHICs isolated via mechanical digestion of the liver were processed according to the 10x Chromium Single-cell V(D)J Reagent Kits User Guide and loaded on to the Chromium 10x platform using the 5' v1.1 chemistry (10x Genomics). Libraries were sequenced on a NovaSeq 6000 platform (PE150) (Illumina) to an average ~50,000 reads per cell.