AAV-HBV mouse model replicates immune exhaustion patterns of chronic HBV patients at single-cell level

Abstract 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 healthy 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. Lay Summary The AAV-HBV mouse model is used as a research tool to study hepatitis B infection. In this study we evaluated the translation value from mouse to human with regards to T-cell exhaustion. Highlights AAV-HBV mice transduced with a high titer vector showed presence of CD8 exhausted T-cells after 24 weeks. High titer transduced mice, but not lower titer show increased expression of LAG-3, TOX, TIM-3 and TIGIT in CD8 T-cells. PD-1 was increased in CD8 T-cells, independent of HBV transduction titer. A similar exhausted CD8 T-cell population could be found in chronic HBV donors, but not in healthy individuals. Graphical abstract