Class A capsid assembly modulator apoptotic elimination of hepatocytes with high HBV core antigen level in vivo is dependent on de novo core protein translation

Abstract

ABSTRACTBackground and aimsCapsid assembly (CA) is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. CA is the target for various new anti-viral candidate therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and lead to hepatocyte cell death. The aim of the studies was to identify the mechanism of action of the CAM-A modulators leading to HBV infected hepatocyte elimination.MethodsThe CAM-A mediated mechanism of HBsAg reduction was evaluated in vitro in a stable HBV replicating cell line and in vivo in AAV-HBV transduced C57BL/6, C57BL/6 SCID and HBV-infected chimeric mice with humanized livers.ResultsIn vivo treatment with CAM-A modulators induced pronounced reductions in HBe- and HBsAg which were associated with a transient increase in ALT. Both HBs- and HBeAg reduction and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute to this phenotype. However, depletion of CD8+ T-cells in transduced wild-type mice did not have a negative impact on antigen reduction, indicating that CD8+ T-cell responses are not essential.Coinciding with the transient ALT elevation in AAV-HBV transduced mice, we observed a transient increase in markers related to endoplasmic reticulum stress and apoptosis as well as cytokines related to apoptosis pathways, followed by the detection of a proliferation marker. Pathway enrichment analysis of microarray data revealed that antigen presentation pathway (MHC-I) was upregulated, overlapping with observed apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A mediated HBsAg reduction is dependent on de novo core protein translation and that the effect is dependent on high levels of core protein expression, which will likely focus the CHB sub-population that could respond.ConclusionCAM-A treatment eradicates HBV infected hepatocytes with high core protein levels through the induction of apoptosis a promising approach as part of a regimen to achieve functional cure.Lay summaryTreatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on thede novosynthesis and high levels of core protein.