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Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure

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Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure. / Allweiss, Lena; Testoni, Barbara; Yu, Mei; Lucifora, Julie; Ko, Chunkyu; Qu, Bingqian; Lütgehetmann, Marc; Guo, Haitao; Urban, Stephan; Fletcher, Simon P; Protzer, Ulrike; Levrero, Massimo; Zoulim, Fabien; Dandri-Petersen, Maura.

In: GUT, Vol. 72, No. 5, 05.2023, p. 972-983.

Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review

Harvard

Allweiss, L, Testoni, B, Yu, M, Lucifora, J, Ko, C, Qu, B, Lütgehetmann, M, Guo, H, Urban, S, Fletcher, SP, Protzer, U, Levrero, M, Zoulim, F & Dandri-Petersen, M 2023, 'Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure', GUT, vol. 72, no. 5, pp. 972-983. https://doi.org/10.1136/gutjnl-2022-328380

APA

Allweiss, L., Testoni, B., Yu, M., Lucifora, J., Ko, C., Qu, B., Lütgehetmann, M., Guo, H., Urban, S., Fletcher, S. P., Protzer, U., Levrero, M., Zoulim, F., & Dandri-Petersen, M. (2023). Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure. GUT, 72(5), 972-983. https://doi.org/10.1136/gutjnl-2022-328380

Vancouver

Allweiss L, Testoni B, Yu M, Lucifora J, Ko C, Qu B et al. Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure. GUT. 2023 May;72(5):972-983. https://doi.org/10.1136/gutjnl-2022-328380

Bibtex

@article{cc2c6310463d4dc9a794a2ee2b1cc7b1,
title = "Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure",
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.",
author = "Lena Allweiss and Barbara Testoni and Mei Yu and Julie Lucifora and Chunkyu Ko and Bingqian Qu and Marc L{\"u}tgehetmann and Haitao Guo and Stephan Urban and Fletcher, {Simon P} and Ulrike Protzer and Massimo Levrero and Fabien Zoulim and Maura Dandri-Petersen",
note = "{\textcopyright} Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.",
year = "2023",
month = may,
doi = "10.1136/gutjnl-2022-328380",
language = "English",
volume = "72",
pages = "972--983",
journal = "GUT",
issn = "0017-5749",
publisher = "BMJ PUBLISHING GROUP",
number = "5",

}

RIS

TY - JOUR

T1 - Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure

AU - Allweiss, Lena

AU - Testoni, Barbara

AU - Yu, Mei

AU - Lucifora, Julie

AU - Ko, Chunkyu

AU - Qu, Bingqian

AU - Lütgehetmann, Marc

AU - Guo, Haitao

AU - Urban, Stephan

AU - Fletcher, Simon P

AU - Protzer, Ulrike

AU - Levrero, Massimo

AU - Zoulim, Fabien

AU - Dandri-Petersen, Maura

N1 - © Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

PY - 2023/5

Y1 - 2023/5

N2 - 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.

AB - 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.

U2 - 10.1136/gutjnl-2022-328380

DO - 10.1136/gutjnl-2022-328380

M3 - SCORING: Journal article

C2 - 36707234

VL - 72

SP - 972

EP - 983

JO - GUT

JF - GUT

SN - 0017-5749

IS - 5

ER -