Proliferation of hepatitis B virus infected human hepatocytes in humanized mice treated with the entry inhibitor Myrcludex-B induces strong cccDNA reduction and maintenance of cccDNA-free hepatocytes

Abstract

In chronic hepatitis B virus (HBV) infection, the viral genome forms a stable minichromosome, the covalently closed circular DNA (cccDNA), which can persist throughout the hepatocyte lifespan. Immune mediated cell injury and compensatory cell growth, however, appear to favor cccDNA destabilization, leading to the selection of cccDNA-free hepatocytes. Aim of the study was to determine the proliferative capacities of HBV-infected human hepatocytes in vivo as well as the impact of liver regeneration on intrahepatic cccDNA loads in human-liver chimeric uPA/SCID mice treated for sixty days with the entry inhibitor Myrcludex-B to avoid de novo infection of cccDNA-cleared hepatocytes. Methods: Human hepatocyte proliferation was triggered by transplanting HBV-infected primary human hepatocytes (PHH) isolated from highly viremic (1xE9 HBV-DNA/ml) humanized mice into naive uPA/SCID recipients. PHH proliferation and viral load changes were determined by qRT-PCR and immunohistochemistry. Results: Isolated PHHs engrafted and strongly proliferated in recipient mice reaching an average of 5 to 6 cell doublings within 100 days. Although all PHHs appeared HBcAg-positive in the donor mouse, HBcAg staining was lost quickly during the first four weeks after transplantation. Notably, HBcAg-positive PHHs displayed a marked reduction in their proliferation capacity compared to HBcAg-negative PHHs as measured by Ki67 co-staining. Not only intrahepatic HBV-RNA (pgRNA and subgenomic RNA) levels decreased in the first 30 days post-transplantation, but also cccDNA copies/PHH dropped by more than 2 log. This was due to both a dilution of the cccDNA pool among daughter cells and to a 1.4-log loss of total intrahepatic cccDNA loads. However, serological and intrahepatic virological markers rebounded at later time points, as PHH expansion relented. This was due to a de novo infection of quiescent hepatocytes because treatment of mice with Myrcludex-B from day 30 – 90 completely blocked viral rebound. Conclusion: Even if cell division appeared to be less pronounced in HBcAg-positive human hepatocytes, in the milieu of a strong liver regeneration, expansion of PHHs induced a strong cccDNA reduction, leading to the formation of cccDNA-free hepatocytes. Since administration of the entry inhibitor efficiently prevented re-infection of quiescent hepatocytes, viral clearance could be achieved in the great majority of chronically infected hepatocytes.