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Original article
Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo
  1. Lena Allweiss1,
  2. Tassilo Volz1,
  3. Katja Giersch1,
  4. Janine Kah1,
  5. Giuseppina Raffa2,
  6. Joerg Petersen3,
  7. Ansgar W Lohse1,4,
  8. Concetta Beninati5,
  9. Teresa Pollicino2,
  10. Stephan Urban4,6,
  11. Marc Lütgehetmann1,7,
  12. Maura Dandri1,4
  1. 1Department of Medicine, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  2. 2Department of Clinical and Experimental Medicine, University Hospital of Messina, Messina, Italy
  3. 3IFI Institute for Interdisciplinary Medicine at Asklepios Clinic St. Georg, Hamburg, Germany
  4. 4German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel and Heidelberg Partner Sites, Hamburg, Germany
  5. 5Department of Human Pathology, University Hospital of Messina, Messina, Italy
  6. 6Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
  7. 7Department of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  1. Correspondence to Professor Dr Maura Dandri, Department of Internal Medicine, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52, Hamburg D-20246, Germany; m.dandri{at}uke.de

Abstract

Objective The stability of the covalently closed circular DNA (cccDNA) in nuclei of non-dividing hepatocytes represents a key determinant of HBV persistence. Contrarily, studies with animal hepadnaviruses indicated that hepatocyte turnover can reduce cccDNA loads but knowledge on the proliferative capacity of HBV-infected primary human hepatocytes (PHHs) in vivo and the fate of cccDNA in dividing PHHs is still lacking. This study aimed to determine the impact of human hepatocyte division on cccDNA stability in vivo.

Methods PHH proliferation was triggered by serially transplanting hepatocytes from HBV-infected humanised mice into naïve recipients. Cell proliferation and virological changes were assessed by quantitative PCR, immunofluorescence and RNA in situ hybridisation. Viral integrations were analysed by gel separation and deep sequencing.

Results PHH proliferation strongly reduced all infection markers, including cccDNA (median 2.4 log/PHH). Remarkably, cell division appeared to cause cccDNA dilution among daughter cells and intrahepatic cccDNA loss. Nevertheless, HBV survived in sporadic non-proliferating human hepatocytes, so that virological markers rebounded as hepatocyte expansion relented. This was due to reinfection of quiescent PHHs since treatment with the entry inhibitor myrcludex-B or nucleoside analogues blocked viral spread and intrahepatic cccDNA accumulation. Viral integrations were detected both in donors and recipient mice but did not appear to contribute to antigen production.

Conclusions We demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies.

  • CHRONIC HEPATITIS
  • HEPATITIS B
  • ANTIVIRAL THERAPY
  • BASIC SCIENCES

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Footnotes

  • Contributors MD, JP and ML initiated and supervised the study. MD, ML and LA designed experiments and analysed data. LA, TV and KG generated chimeric mice and performed animal experiments. LA performed hepatocyte isolations and molecular analyses. SU provided the myrcludex-B. TP and GR performed the Alu-PCR. CB performed the deep sequencing. JK performed the Luminex analysis. LA and MD wrote the manuscript. JP, ML, TV, TP, SU and AWL discussed the data and corrected the manuscript.

  • Funding The study was supported by the German Research Foundation (MD Heisenberg Professorship DA1063/3-2; Collaborative Research Centre SFB-841: A5). MD and SU also receive funding from the German Center for Infection Research (DZIF; TTU05.806 and TTU05.704).

  • Competing interests SU is co-applicant and inventor of patents protecting myrcludex-B.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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