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Journal Article•DOI•

Persistence of cccDNA During the Natural History of Chronic Hepatitis B and Decline During Adefovir Dipivoxil Therapy

Bettina Werle-Lapostolle1, Scott Bowden, Stephen Locarnini, Karsten Wursthorn2, Karsten Wursthorn3, Jörg Petersen2, Jörg Petersen3, George K. K. Lau4, Christian Trepo, Patrick Marcellin, Zachary Goodman5, William E. Delaney, Shelly Xiong, Carol L. Brosgart, Shan-Shan Chen, Craig S. Gibbs, Fabien Zoulim •
French Institute of Health and Medical Research1, University of Hamburg2, Heinrich Pette Institute3, Queen Mary Hospital4, Armed Forces Institute of Pathology5
01 Jun 2004-Gastroenterology (Gastroenterology)-Vol. 126, Iss: 7, pp 1750-1758
TL;DR: cccDNA persists throughout the natural history of chronic hepatitis B, even in patients with serologic evidence of viral clearance, and long-term ADV therapy significantly decreased cccDNA levels by a primarily noncytolytic mechanism.
Abstract: BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a unique episomal replicative intermediate responsible for persistent infection of hepatocytes. Technical constraints have hampered the direct study of cccDNA maintenance and clearance mechanisms in patients. The aim of this study was to develop a sensitive and specific assay for quantifying cccDNA in biopsy samples from chronic hepatitis B patients during different natural history phases and in patients undergoing antiviral therapy. METHODS: Intrahepatic cccDNA levels were quantified by a specific real-time PCR assay. Ninety-eight liver biopsy samples from patients in the major phases of the natural history of chronic hepatitis B and 32 pairs of samples from patients receiving adefovir dipivoxil (ADV) therapy were assessed. RESULTS: cccDNA was detected, at levels ranging over 3 orders of magnitude, in patients in different phases of the natural history of chronic hepatitis B. cccDNA levels were strongly correlated with levels of total intracellular HBV DNA and serum HBV DNA. Forty-eight weeks of ADV therapy resulted in a significant 0.8 log decrease in cccDNA copies/cell. Changes in cccDNA were correlated with a similar reduction in serum HBsAg titer but not with a decrease in the number of HBV antigen-positive cells during ADV treatment.CONCLUSIONS: cccDNA persists throughout the natural history of chronic hepatitis B, even in patients with serologic evidence of viral clearance. Long-term ADV therapy significantly decreased cccDNA levels by a primarily noncytolytic mechanism.

Summary (1 min read)

Jump to: [Statistical analysis] and [Figure Legends]

Statistical analysis

  • Statistical comparisons were performed using SAS v8.1 (SAS Institute Cary, NC USA).
  • Two-group comparisons were performed using Wilcoxon rank-sum tests; comparisons of more than 2 groups were made using Kruskal-Wallis tests.
  • Changes in cccDNA were highly correlated with changes in total intrahepatic HBV DNA (P<0.001) as well as with changes in serum HBV DNA (P=0.008).
  • Importantly, reductions in cccDNA were correlated with, and similar in magnitude to, reductions in serum HBsAg titer.
  • This also suggests that the clearance of cccDNA during antiviral therapy may be occurring primarily through a mechanism other than infected cell killing.

Figure Legends

  • Figure 1. Design and validation of a selective assay for HBV cccDNA amplification.
  • A, PCR primers for amplification of cccDNA are targeted to opposite sides of the single-stranded gap regions of relaxed circular (RC) HBV DNA.
  • Each point represent a single patient with the bars indicating median values.
  • B, Change in total intracellular HBV DNA levels between baseline and week 48 in 22 ADV-treated patients and 10 PBO patients, integrated results.
  • D, Change in cccDNA levels between baseline and week 48 in ADV-treated and placebo patients as reported by individual analysis sites (Lyon, Hamburg, Melbourne), bars represent median values and error bars indicate interquartile ranges.

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Citations
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Journal Article•DOI•
Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus

[...]

Huan Yan1, Guocai Zhong, Guangwei Xu, Wenhui He2, Zhiyi Jing, Zhenchao Gao1, Yi Huang2, Yonghe Qi, Bo Peng, Haimin Wang, Liran Fu2, Mei Song2, Pan Chen2, Wenqing Gao, Bijie Ren, Yinyan Sun, Tao Cai, Xiaofeng Feng, Jianhua Sui, Wenhui Li •
Peking University1, Peking Union Medical College2
13 Nov 2012-eLife
TL;DR: It is shown that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver that is a functional receptor for HBV and HDV.
Abstract: Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.

1,662 citations

Cites methods from "Persistence of cccDNA During the Na..."

  • ...HBV cccDNA (double-stranded DNA without nick and gap) was quantified by real-time PCR using a protocol as previously described Werle-Lapostolle et al., 2004) with modifications....

    [...]

  • ...HBV cccDNA (double-stranded DNA without nick and gap) was quantified by real-time PCR using a protocol as previously described (Bowden et al., 2004; Werle-Lapostolle et al., 2004) with modifications....

    [...]

Journal Article•DOI•
Statements from the Taormina expert meeting on occult hepatitis B virus infection.

[...]

Giovanni Raimondo1, Jean-Pierre Allain2, Maurizia Rossana Brunetto, Marie Annick Buendia3, Ding-Shinn Chen4, Massimo Colombo5, Antonio Craxì6, Francesco Donato7, Carlo Ferrari, Giovanni Battista Gaeta8, Wolfram H. Gerlich9, Massimo Levrero10, Stephen Locarnini, Thomas I. Michalak11, Mario U. Mondelli12, Jean-Michel Pawlotsky13, Teresa Pollicino1, Daniele Prati5, Massimo Puoti7, Didier Samuel14, Daniel Shouval15, Antonina Smedile, Giovanni Squadrito1, Christian Trepo16, Erica Villa17, Hans Will18, Alessandro Zanetti5, Fabien Zoulim16 •
University of Messina1, University of Cambridge2, Pasteur Institute3, National Taiwan University4, University of Milan5, University of Palermo6, University of Brescia7, Seconda Università degli Studi di Napoli8, University of Giessen9, Sapienza University of Rome10, St. John's University11, University of Pavia12, University of Paris13, University of Paris-Sud14, Hebrew University of Jerusalem15, Claude Bernard University Lyon 116, University of Modena and Reggio Emilia17, University of Hamburg18
01 Oct 2008-Journal of Hepatology
TL;DR: Giovanni Raimondo*, Jean-Pierre Allain, Maurizia R. Brunetto, Marie-Annick Buendia, Ding-Shinn Chen, Massimo Colombo, Antonio Craxi, Francesco Donato, Carlo Ferrari, Giovanni B. Gaeta, Wolfram H. Gerlich,Massimo Levrero, Stephen Locarnini, Thomas Michalak, Mario U. Zanetti, Fabien Zoulim
Abstract: Giovanni Raimondo*, Jean-Pierre Allain, Maurizia R. Brunetto, Marie-Annick Buendia, Ding-Shinn Chen, Massimo Colombo, Antonio Craxi, Francesco Donato, Carlo Ferrari, Giovanni B. Gaeta, Wolfram H. Gerlich, Massimo Levrero, Stephen Locarnini, Thomas Michalak, Mario U. Mondelli, Jean-Michel Pawlotsky, Teresa Pollicino, Daniele Prati, Massimo Puoti, Didier Samuel, Daniel Shouval, Antonina Smedile, Giovanni Squadrito, Christian Trepo, Erica Villa, Hans Will, Alessandro R. Zanetti, Fabien Zoulim

740 citations

Cites background from "Persistence of cccDNA During the Na..."

  • ...The molecular basis of persistent OBI is related to the long-lasting persistence of HBV cccDNA in the nuclei of hepatocytes [4,26,41–45]....

    [...]

Journal Article•DOI•
Hepatitis B virus resistance to nucleos(t)ide analogues.

[...]

Fabien Zoulim1, Fabien Zoulim2, Stephen Locarnini•
University of Lyon1, French Institute of Health and Medical Research2
01 Nov 2009-Gastroenterology
TL;DR: It is important to continue basic research into HBV replication and pathogenic mechanisms to identify new therapeutic targets, develop novel antiviral agents, design combination therapies that prevent drug resistance, and decrease the incidence of complications of CHB.
Abstract: Patients with chronic hepatitis B (CHB) can be successfully treated using nucleos(t)ide analogs (NA), but drug-resistant hepatitis B virus (HBV) mutants frequently arise, leading to treatment failure and progression to liver disease. There has been much research into the mechanisms of resistance to NA and selection of these mutants. Five NA have been approved by the US Food and Drug Administration for treatment of CHB; it is unlikely that any more NA will be developed in the near future, so it is important to better understand mechanisms of cross-resistance (when a mutation that mediates resistance to one NA also confers resistance to another) and design more effective therapeutic strategies for these 5 agents. The genes that encode the polymerase and envelope proteins of HBV overlap, so resistance mutations in polymerase usually affect the hepatitis B surface antigen; these alterations affect infectivity, vaccine efficacy, pathogenesis of liver disease, and transmission throughout the population. Associations between HBV genotype and resistance phenotype have allowed cross-resistance profiles to be determined for many commonly detected mutants, so genotyping assays can be used to adapt therapy. Patients that experience virologic breakthrough or partial response to their primary therapy can often be successfully treated with a second NA, if this drug is given at early stages of these events. However, best strategies for preventing NA resistance include first-line use of the most potent antivirals with a high barrier to resistance. It is important to continue basic research into HBV replication and pathogenic mechanisms to identify new therapeutic targets, develop novel antiviral agents, design combination therapies that prevent drug resistance, and decrease the incidence of complications of CHB.

659 citations

Journal Article•DOI•
HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B

[...]

Michael Nassal
03 Jun 2015-Gut
TL;DR: This review aims to summarise current knowledge on ccc DNA molecular biology, to highlight the experimental restrictions that have hitherto hampered faster progress and to discuss cccDNA as target for new, potentially curative therapies of chronic hepatitis B.
Abstract: At least 250 million people worldwide are chronically infected with HBV, a small hepatotropic DNA virus that replicates through reverse transcription. Chronic infection greatly increases the risk for terminal liver disease. Current therapies rarely achieve a cure due to the refractory nature of an intracellular viral replication intermediate termed covalently closed circular (ccc) DNA. Upon infection, cccDNA is generated as a plasmid-like episome in the host cell nucleus from the protein-linked relaxed circular (RC) DNA genome in incoming virions. Its fundamental role is that as template for all viral RNAs, and in consequence new virions. Biosynthesis of RC-DNA by reverse transcription of the viral pregenomic RNA is now understood in considerable detail, yet conversion of RC-DNA to cccDNA is still obscure, foremostly due to the lack of feasible, cccDNA-dependent assay systems. Conceptual and recent experimental data link cccDNA formation to cellular DNA repair, which is increasingly appreciated as a critical interface between cells and viruses. Together with new in vitro HBV infection systems, based on the identification of the bile acid transporter sodium taurocholate cotransporting polypeptide as an HBV entry receptor, this offers novel opportunities to decipher, and eventually interfere with, formation of the HBV persistence reservoir. After a brief overview of the role of cccDNA in the HBV infectious cycle, this review aims to summarise current knowledge on cccDNA molecular biology, to highlight the experimental restrictions that have hitherto hampered faster progress and to discuss cccDNA as target for new, potentially curative therapies of chronic hepatitis B.

632 citations

Journal Article•DOI•
Occult hepatitis B virus infection

[...]

Giovanni Raimondo1, Teresa Pollicino1, Irene Cacciola1, Giovanni Squadrito1•
University of Messina1
01 Jan 2007-Journal of Hepatology
TL;DR: Much evidence suggests that hepatitis B virus can favour the progression of liver fibrosis and above all the development of hepatocellular carcinoma, and is an entity with world-wide diffusion.
Abstract: The persistence of hepatitis B virus (HBV) genomes in HBV surface antigen (HBsAg) negative individuals is termed occult HBV infection. Occult HBV status is associated in some cases with mutant viruses undetectable by HBsAg assays, but more frequently it is due to a strong suppression of viral replication and gene expression. Occult HBV infection is an entity with world-wide diffusion, although the available data of prevalence in various categories of individuals are often contrasting because of the different sensitivity and specificity of the methods used for its detection in many studies. Occult HBV may impact in several different clinical contexts, including the transmission of the infection by blood transfusion or organ transplantation and its acute reactivation when an immunosuppressive status occurs. Moreover, much evidence suggests that it can favour the progression of liver fibrosis and above all the development of hepatocellular carcinoma.

608 citations

Cites result from "Persistence of cccDNA During the Na..."

  • ...In fact, the quantification of intra-hepatic viral DNA in different sets of chronic HBV infected individuals demonstrated that the amount of HBV DNA in the liver of occult HBV infected patients is often comparable to that detected in HBsAg positive individuals [51]....

    [...]

References
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Journal Article•DOI•
Identification of a Reservoir for HIV-1 in Patients on Highly Active Antiretroviral Therapy

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Diana Finzi1, Monika Hermankova, Theodore C. Pierson, Lucy M. Carruth, Christopher B. Buck, Richard E. Chaisson, Thomas C. Quinn, Karen Chadwick, Joseph B. Margolick, Ron Brookmeyer, Joel E. Gallant, Martin Markowitz, David D. Ho, Douglas D. Richman, Robert F. Siliciano •
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Daniel Abramowicz1, Cédric Blanpain1, Christiane Knoop1•
Autonomous University of Madrid1
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William M. Lee
11 Dec 1997-The New England Journal of Medicine
TL;DR: This review addresses many aspects of HBV infection, including the role of the immune system in determining the outcome of clinical infection, recent developments in molecular studies of the virus, and new treatments capable of eradicating chronic infection.
Abstract: The hepatitis B virus (HBV), discovered in 1966, infects more than 350 million people worldwide.1 Hepatitis B is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma, accounting for 1 million deaths annually. Knowledge of the intricacies of viral infection and of the molecular biology of this fascinating virus has led to the successful development of a vaccine and to treatment sometimes capable of eradicating chronic infection. This review addresses many aspects of HBV infection, including the role of the immune system in determining the outcome of clinical infection, recent developments in molecular studies of the virus, and new . . .

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Journal Article•DOI•
Chronic hepatitis B

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Anna S. Lok1, Brian J. McMahon2•
University of Michigan1, Alaska Native Medical Center2
01 Dec 2001-Hepatology
TL;DR: These guidelines have been written to assist physicians and other health care providers in the recognition, diagnosis, and management of patients chronically infected with the hepatitis B virus (HBV).
Abstract: PREAMBLE These guidelines have been written to assist physicians and other health care providers in the recognition, diagnosis, and management of patients chronically infected with the hepatitis B virus (HBV). They are intended to suggest preferable approaches to the clinical management of chronic hepatitis B. The recommendations are flexible and are not intended as the only acceptable approach to management and treatment. As the appropriate course of treatment will vary in light of the relevant facts and circumstances surrounding each individual patient with chronic hepatitis B, guidelines are not intended to define the applicable standard of medical care and may be updated periodically as new information becomes available. These guidelines were developed under the auspices of, and approved by, the Practice Guidelines Committee of the American Association for the Study of Liver Diseases. They should be taken as guidelines and not “standards of care.” Data used to support the recommendations made were obtained by a literature search of peer-reviewed articles concerning the natural history, diagnosis, and treatment of chronic hepatitis B. In addition, the proceedings of a recent National Institutes of Health workshop on the “Management of Hepatitis B” were considered in the development of these guidelines. 1 The strength of each recommendation is categorized based on the quality of evidence in the literature according to the rating system indicated in Table 1. 2

2,790 citations

Journal Article•DOI•
Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy

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Tae-Wook Chun1, Lieven Stuyver, Stephanie B. Mizell, Linda A. Ehler, Jo Ann M. Mican, Michael Baseler, Alun L. Lloyd, Martin A. Nowak, Anthony S. Fauci •
National Institutes of Health1
25 Nov 1997-Proceedings of the National Academy of Sciences of the United States of America
TL;DR: Highly purified CD4+ T cells from patients receiving HAART with an average treatment time of 10 months and with undetectable plasma viremia carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro, suggesting persistent active virus replication in vivo.
Abstract: Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.

1,834 citations

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Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "Persistence of cccdna during the natural history of chronic hepatitis b and decline during adefovir dipivoxil therapy" ?

The aim of this study was to develop a sensitive and specific assay quantifying cccDNA in biopsy samples from chronic hepatitis B patients during different natural history phases and in patients undergoing antiviral therapy.