Sustained liver HBsAg loss and clonal T and B cell expansion upon therapeutic DNA vaccination require low HBsAg levels

Background & Aims Suppression of HBV DNA, inhibition of HBsAg production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBsAg when hepatitis B surface (HBsAg) levels are low. The factor(s) required for mounting an effective immune control of HBV infection are unclear. Using a single-cell approach, we investigated the liver immune environment in the context of different levels of HBsAg as well as upon sustained HBsAg loss through treatment with an HBV specific GalNAc-siRNA followed by therapeutic vaccination. Methods C57BL/6 mice were transduced with a range of rAAV-HBV DNA to express different HBsAg levels. Mice were treated with GalNAc-siRNA targeting HBV transcripts to lower the HBsAg levels and then vaccinated 4 times with a DNA vaccine encoding HBV Core, Pol and Surface. We used single-cell RNA-sequencing on homogenised liver resident cells, paired with single-cell V(D)J receptor sequencing to understand the changes in the liver immune microenvironment. Results Treatment with GalNAc-HBV siRNA followed by therapeutic vaccination, achieved a sustained HBsAg loss in all mice. This was accompanied by an induction of CD4 follicular helper T-cell responses, polyclonal activation of CD8 T-cells in the liver and clonal expansion of plasma cells that were responsible for antibody production. Conclusions This study provides novel insight into the immune changes in the liver at the single-cell level, highlighting the correlation between the induced reduction in HBsAg levels and the clonal expansion of CD4 follicular helper T-cells, CD8 cytotoxic T-cells, plasma cells, and ISG-producing neutrophils in the liver upon HBV siRNA and subsequent therapeutic vaccine treatment. Lay Summary Chronic hepatitis B infection is characterized by a complex interplay between immune responses and viral replication in the liver. To achieve functional cure a combination of different treatments is likely required. In this study single-cell approach was used to understand the liver microenvironment in the context of different HBsAg levels followed by therapeutic vaccination in AAV-HBV mouse model and to identify key factors required to achieve functional cure. ![Figure][1] Highlights ### Competing Interest Statement Authors that are employees of Janssen Research and Development, may be Johnson & Johnson stockholders. * AAV : adeno-associated virus BCR : B cell receptor CDC : constitutive dendritic cell CHB : chronic hepatitis B GalNAc : N-acetylgalactosamine HBV : hepatitis B virus HbsAg : hepatitis B surface antigen HbeAg : hepatitis B e antigen HbsAb : hepatitis B surface antibody HBcAg : hepatitis B Core IHC : immunohistochemistry IHL : intrahepatic lymphocytes ISG : interferon stimulating genes LSEC : Liver sinusoidal endothelial cells MVA : modified vaccinia Ankara NK-cell : natural killer cell PDC : plasmacytoid dendritic cell TCR : T-cell receptor Tfh : T follicular helper cell UMAP : Uniform Manifold Approximation and Projection UMI : Unique Molecular Identifier vge : viral genome equivalents WHO : World Health Organization [1]: pending:yes