High antigen burden drives CD8+ T cell dysfunction in a mouse model of chronic hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is characterized by dysfunctional HBV-specific CD8+ T cell responses that contribute to viral persistence. Understanding the mechanisms behind this dysfunction is necessary to develop effective immunotherapies. Here, we used the adeno-associated virus (AAV)-HBV mouse model of HBV replication to investigate the development of CD8+ T cell dysfunction during chronic HBV infection. Unlike naïve mice, AAV-HBV mice with intermediate to high viral antigen levels failed to respond to therapeutic immunization against HBV antigens, indicating that failure to initially induce HBV-specific CD8+ T cell responses cannot account for the entirety of the observed CD8+ T cell dysfunction. Similarly, mice challenged with a high dose of AAV-HBV could not control antigenemia despite initially functional CD8+ T cell responses generated by immunization. These mice exhibited rapid CD8+ T cell functional impairment, marked by reduced cytokine production and increased expression of the inhibitory receptor PD-1. In contrast, mice challenged with a lower dose of AAV-HBV effectively controlled viral antigens and maintained CD8+ T cell function. Combination treatment with therapeutic immunization and antibody-mediated PD-1 and CTLA-4 blockade partially reduced HBsAg levels in mice with intermediate but not higher antigen burdens. These findings indicate that during chronic HBV infection, high antigen expression is an important factor driving a CD8+ T cell dysfunction that cannot be easily reversed and may limit the efficacy of some immunotherapeutic approaches.IMPORTANCEDespite available preventative vaccines, chronic hepatitis B virus (HBV) infection remains a major cause of liver disease worldwide, with no adequate cure. Challenges in developing effective immunotherapies include our incomplete understanding of immune dysfunction mechanisms and limited representative animal models. Here, we further characterized CD8+ T cell dysfunction mechanisms in the AAV-HBV mouse model, which shares some immunological features with human chronic HBV infection (CHB). We observed that high but not low antigen levels induced CD8+ T cell dysfunction marked by elevated PD-1 expression and led to viral persistence, despite HBsAg clearance. Additionally, responsiveness to combination immunotherapies was influenced by antigen levels at the time of treatment. This work illustrates the interplay between antigen load, immune checkpoints, and immune tolerance during CHB and may offer insights into potential strategies for enhancing HBV-specific immune responses to promote a functional cure.