Author information

• ¹I. Medical Clinic and Polyclinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
• ²German Center for Infection Research, Hamburg-Lübeck-Borstel-Riems, Munich and Heidelberg sites, Germany.
• ³Cancer Research Center of Lyon, INSERM U1052, Lyon University, Hospices de Lyon, Lyon, France.
• ⁴ANRS HBV Cure Task Force, Lyon, France.
• ⁵Gilead Sciences, Foster City, California, USA.
• ⁶CIRI, Centre International de Recherche en Infectiologie, INSERM U1111, Université Claude Bernard Lyon 1, Lyon, France.
• ⁷Institute of Virology, Technical University of Munich, Munchen, Germany.
• ⁸Infectious Diseases Therapeutic Research Center, Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, Korea (the Republic of).
• ⁹Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany.
• ¹⁰Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany.
• ¹¹Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf Hamburg, Hamburg, Germany.
• ¹²Indiana University School of Medicine, Indianapolis, Indiana, USA.
• ¹³Department of Microbiology and Molecular Genetics, Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
• ¹⁴Cancer Research Center of Lyon, INSERM U1052, Lyon University, Hospices de Lyon, Lyon, France m.dandri@uke.de fabien.zoulim@inserm.fr.
• ¹⁵I. Medical Clinic and Polyclinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany m.dandri@uke.de fabien.zoulim@inserm.fr.

Abstract

Objectives: A major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices.

Design: Reference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/-PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB.

Results: Hirt and -PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA.

Conclusions: We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.