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Singh R, Ramakrishna G, Sharma MK, Kumar R, Gupta E, Rastogi A, Tanwar P, Sarin SK, Trehanpati N. Droplet digital PCR technique is ultrasensitive for the quantification of covalently closed circular DNA in the blood of chronic HBV-infected patients. Clin Res Hepatol Gastroenterol 2025; 49:102531. [PMID: 39832728 DOI: 10.1016/j.clinre.2025.102531] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/14/2024] [Revised: 01/13/2025] [Accepted: 01/17/2025] [Indexed: 01/22/2025]
Abstract
BACKGROUND Covalently closed circular DNA (cccDNA) is a stable, episomal form of HBV DNA. cccDNA is a true marker for the intrahepatic events in controlled CHB infection. Quantifying cccDNA is critical for monitoring disease progression, and efficacy of anti-viral therapies. METHODS To standardize the method, total HBV DNA was isolated from HepAD38 cells and digested with three exonuclease enzymes to remove linear and relaxed circular HBV DNA. Purified cccDNA quantification used ddPCR with specific primers. Treatment-naive chronic hepatitis B virus patients (nCHBV, n=36) with detectable HBV DNA and HBsAg were grouped by HBsAg levels: Group I (HBsAglo < 2000 IU/ml, n=11) and Group II (HBsAghi > 2000 IU/ml, n=25). cccDNA, HBV DNA and HBsAg were quantified in plasma and compared between groups. Correlation with clinical/histopathological features was done. RESULTS Non-digested 3.6^10⁶ tet-ve HepAD38 cells showed 316 copies/µl of total viral DNA. After digesting the linear, integrated, and relaxed circular DNA with triple enzymes, 15 copies/µl of cccDNA were detected. Similarly, after DNA digestion, HBsAglo patients showed a median of 8.5 copies/µl (IQR 2.75-9.75 copies/µl), and HBsAghi gave a median of 11 copies/µl (IQR 4-16 copies/µl) but with no significant difference between groups (p=0.093). Further, HBsAglo patients with low cccDNA copy numbers showed significantly higher fibrosis grades than HBsAghi (p=0.036). CONCLUSIONS We conclude that employing a combined approach utilizing three exonucleases, cccDNA-specific primers, and ddPCR enables the detection of cccDNA copies even in patients exhibiting low levels of HBsAg and HBV DNA. This integrated method offers additional validation as a surrogate diagnostic tool.
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Affiliation(s)
- Ravinder Singh
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Gayatri Ramakrishna
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Manoj Kumar Sharma
- Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Rahul Kumar
- Laboratory Oncology Unit, Dr. B. R. A. Institute Rotary Cancer Hospital (Cancer Centre), All India Institute of Medical Sciences, New Delhi, India
| | - Ekta Gupta
- Department of Virology, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Archana Rastogi
- Department of Pathology, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Pranay Tanwar
- Laboratory Oncology Unit, Dr. B. R. A. Institute Rotary Cancer Hospital (Cancer Centre), All India Institute of Medical Sciences, New Delhi, India
| | - Shiv Kumar Sarin
- Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India.
| | - Nirupama Trehanpati
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India.
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Lumley SF, Kent C, Jennings D, Chai H, Airey G, Waddilove E, Delphin M, Trebes A, McNaughton AL, Mohammed KS, Wilkinson SAJ, Wu Y, MacIntyre-Cockett G, Kimono B, Mbonye KM, Ojambo K, Maponga TG, Tan CCS, de Lara C, Martin J, Campbell J, Van Schalkwyk M, Goedhals D, Newton R, Barnes E, Loman NJ, Piazza P, Quick J, Ansari MA, Matthews PC. Whole genome sequencing of hepatitis B virus using tiled amplicon (HEPTILE) and probe based enrichment on Illumina and Nanopore platforms. Sci Rep 2025; 15:5795. [PMID: 39962085 PMCID: PMC11832747 DOI: 10.1038/s41598-025-87721-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2024] [Accepted: 01/21/2025] [Indexed: 02/20/2025] Open
Abstract
Hepatitis B virus (HBV) whole genome sequencing (WGS) is currently limited as the DNA viral loads (VL) of many clinical samples are below the threshold required to generate full genomes using current sequencing methods. We developed two pan-genotypic viral enrichment methods, using probe-based capture and tiled amplicon PCR (HEP-TILE) for HBV WGS. We demonstrate using mock samples that both enrichment methods are pan-genotypic (genotypes A-J). Using clinical samples, we demonstrate that HEP-TILE amplification successfully amplifies full genomes at the lowest HBV VL tested (30 IU/ml), and the PCR products can be sequenced using both Nanopore and Illumina platforms. Probe-based capture with Illumina sequencing required VL > 300,000 IU/ml to generate full length HBV genomes. The capture-Illumina and HEP-TILE-Nanopore pipelines had consensus sequencing accuracy of 100% in mock samples with known DNA sequences. Together, these protocols will facilitate the generation of HBV sequence data, enabling a more accurate and representative picture of HBV molecular epidemiology, cast light on persistence and pathogenesis, and enhance understanding of the outcomes of infection and its treatment.
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Affiliation(s)
- Sheila F Lumley
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
- Department of Infectious Diseases and Microbiology, Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Oxford, UK
| | - Chris Kent
- Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK
| | - Daisy Jennings
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Haiting Chai
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - George Airey
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | | | | | - Amy Trebes
- Nuffield Department of Medicine, Centre for Human Genomics, University of Oxford, Oxford, UK
- Genewiz UK Ltd, Azenta Life Sciences, Stratton Court, Abingdon, UK
| | - Anna L McNaughton
- Population Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK
| | | | - Sam A J Wilkinson
- Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK
| | - Yanxia Wu
- Nuffield Department of Medicine, Centre for Human Genomics, University of Oxford, Oxford, UK
| | | | | | | | - Kevin Ojambo
- MRC/UVRI & LSHTM Uganda Research Unit, Entebbe, Uganda
| | - Tongai G Maponga
- Division of Medical Virology, Stellenbosch University Faculty of Medicine and Health Sciences and National Health Laboratory Service Tygerberg Business Unit, Cape Town, South Africa
| | | | | | - Jacqueline Martin
- Department of Infectious Diseases and Microbiology, Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Oxford, UK
| | | | - Marije Van Schalkwyk
- Division of Infectious Diseases, Department of Medicine, Stellenbosch University and Tygerberg Hospital, Cape Town, South Africa
| | - Dominique Goedhals
- University of the Free State, Bloemfontein, South Africa
- PathCare, Pretoria, South Africa
| | - Robert Newton
- MRC/UVRI & LSHTM Uganda Research Unit, Entebbe, Uganda
- Department of Health Sciences, University of York, York, UK
| | - Eleanor Barnes
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Nicholas J Loman
- Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK
| | - Paolo Piazza
- Nuffield Department of Medicine, Centre for Human Genomics, University of Oxford, Oxford, UK
| | - Joshua Quick
- Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK
| | - M Azim Ansari
- Nuffield Department of Medicine, University of Oxford, Oxford, UK.
| | - Philippa C Matthews
- The Francis Crick Institute, London, UK.
- Division of Infection and Immunity, University College London, London, UK.
- Department of Infection, University College London Hospitals, London, UK.
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Ren EC, Zhuo NZ, Goh ZY, Bonne I, Malleret B, Ko HL. cccDNA-Targeted Drug Screen Reveals a Class of Antihistamines as Suppressors of HBV Genome Levels. Biomolecules 2023; 13:1438. [PMID: 37892121 PMCID: PMC10604930 DOI: 10.3390/biom13101438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Revised: 09/08/2023] [Accepted: 09/22/2023] [Indexed: 10/29/2023] Open
Abstract
Chronic infection with hepatitis B virus (HBV) is incurable, as the current therapeutics cannot eliminate its persistent genomic material, cccDNA. Screening systems for cccDNA-targeting therapeutics are unavailable, as low copies of cccDNA in vitro complicate detection. To address this, cccDNA copies were massively increased to levels detectable via automated plate readers. This was achieved via continuous infection in a contact-free co-culture of an HBV generator (clone F881), which stably produced clinically relevant amounts of HBV, and HBV acceptors selected to carry high cccDNA loads. cccDNA-targeted therapeutics were then identified via reduced cccDNA-specific fluorescence, taking differences in the cell numbers and viability into account. Amongst the drugs tested, the H1 antihistamine Bilastine, HBVCP inhibitors and, surprisingly, current HBV therapeutics downregulated the cccDNA significantly, reflecting the assay's accuracy and sensitivity in identifying drugs that induce subtle changes in cccDNA levels, which take years to manifest in vivo. Bilastine was the only therapeutic that did not reduce HBV production from F881, indicating it to be a novel direct suppressor of cccDNA levels. When further assessed, only the structurally similar antihistamines Pitolisant and Nizatidine suppressed cccDNA levels when other H1 antihistamines could not. Taken together, our rapid fluorescence cccDNA-targeted drug screen successfully identified a class of molecules with the potential to treat hepatitis B.
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Affiliation(s)
- Ee Chee Ren
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
- Immunology Translational Research Programme, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Block MD4, Level 3, Singapore 117545, Singapore;
| | - Nicole Ziyi Zhuo
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
| | - Zhi Yi Goh
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
- Immunology Translational Research Programme, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Block MD4, Level 3, Singapore 117545, Singapore;
| | - Isabelle Bonne
- Immunology Translational Research Programme, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Block MD4, Level 3, Singapore 117545, Singapore;
- Electron Microscopy Unit, Yong Loo Lin School of Medicine, National University of Singapore, MD1, Tahir Foundation Building, #B1-01, 12 Science Drive 2, Singapore 117549, Singapore
- Immunology Programme, Life Sciences Institute, Center for Life Sciences, National University of Singapore, #05-02, 28 Medical Drive, Singapore 117456, Singapore
| | - Benoît Malleret
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
- Immunology Translational Research Programme, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Block MD4, Level 3, Singapore 117545, Singapore;
- Electron Microscopy Unit, Yong Loo Lin School of Medicine, National University of Singapore, MD1, Tahir Foundation Building, #B1-01, 12 Science Drive 2, Singapore 117549, Singapore
| | - Hui Ling Ko
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
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Detectability of Hepatitis B Virus in Peripheral Blood Mononuclear Cells Among Naive Chronic Hepatitis B Patients With Negative Viremia. INFECTIOUS DISEASES IN CLINICAL PRACTICE 2022. [DOI: 10.1097/ipc.0000000000001153] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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DNA Repair Factor Poly(ADP-Ribose) Polymerase 1 Is a Proviral Factor in Hepatitis B Virus Covalently Closed Circular DNA Formation. J Virol 2022; 96:e0058522. [DOI: 10.1128/jvi.00585-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
The biogenesis and eradication of HBV cccDNA have been a research priority in recent years. In this study, we identified the DNA repair factor PARP1 as a host factor required for the HBV
de novo
cccDNA formation.
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Laras A, Papatheodoridi M, Panopoulou E, Papatheodoridis GV, Hadziyannis SJ, Hadziyannis E. Serum hepatitis B virus RNA detectability, composition and clinical significance in patients with ab initio hepatitis B e antigen negative chronic hepatitis B. Virol J 2022; 19:22. [PMID: 35093105 PMCID: PMC8800272 DOI: 10.1186/s12985-022-01749-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Accepted: 01/19/2022] [Indexed: 12/15/2022] Open
Abstract
Background Serum hepatitis B virus (HBV) RNA is a surrogate biomarker for intrahepatic covalently closed circular DNA (cccDNA) transcriptional activity and persistence. In this retrospective study, we investigated its presence, levels and composition in ab initio Hepatitis B e antigen (HBeAg) negative chronically infected patients and examined possible associations with disease activity and the outcome of nucleos(t)ide analogue (NA) discontinuation. Methods We developed a sensitive real time polymerase chain reaction (RT-PCR) for the specific detection of HBV pregenomic RNA (pgRNA) and precore (preC) mRNA and analyzed 220 serum specimens, 160 under NA treatment, from 116 Greek patients initially negative for HBeAg. Results HBV pgRNA was detected in 31% and preC mRNA in 15% of samples, at lower levels representing a small fraction (3.4%) of total core promoter produced transcripts. In the absence of NAs, pgRNA was detected in 57% of samples with median value of 5.19 (2.61–8.35) log10 cp/mL, at lower levels than HBV DNA and correlated significantly with ALT (r = 0.764) and serum HBV DNA (r = 0.906). A wide range of HBV DNA/pgRNA ratio was observed with significant inter- and intra-patient variation. During NA treatment, pgRNA displayed low detectability (22%) and variable levels, median 3.97 (2.30– 8.13) log10 cp/mL, as well as, a significant inverse correlation with the duration of treatment (r = − 0.346, p < 0.01). In 74 events of NA discontinuation, end-of-treatment pgRNA-positive compared to pgRNA-negative cases, experienced more frequently virological (p = 0.016) and clinical (p = 0.011) relapse. Conclusions In genotype D ab initio HBeAg negative patients, serum HBV RNA is primarily composed of pgRNA plus a minor fraction of preC mRNA transcripts. Serum pgRNA is associated with disease activity, suggesting lysis of infected hepatocytes as a possible source of serum HBV RNA in untreated patients and in the early phase of NA treatment. During long term NA treatment, detectable serum pgRNA predicts viral rebound and clinical relapse following treatment discontinuation and may thus serve as a marker for the decision of cessation of therapy.
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7
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Goh ZY, Ren EC, Ko HL. Intracellular interferon signalling pathways as potential regulators of covalently closed circular DNA in the treatment of chronic hepatitis B. World J Gastroenterol 2021; 27:1369-1391. [PMID: 33911462 PMCID: PMC8047536 DOI: 10.3748/wjg.v27.i14.1369] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/21/2021] [Revised: 02/23/2021] [Accepted: 03/17/2021] [Indexed: 02/06/2023] Open
Abstract
Infection with the hepatitis B virus (HBV) is still a major global health threat as 250 million people worldwide continue to be chronically infected with the virus. While patients may be treated with nucleoside/nucleotide analogues, this only suppresses HBV titre to sub-detection levels without eliminating the persistent HBV covalently closed circular DNA (cccDNA) genome. As a result, HBV infection cannot be cured, and the virus reactivates when conditions are favorable. Interferons (IFNs) are cytokines known to induce powerful antiviral mechanisms that clear viruses from infected cells. They have been shown to induce cccDNA clearance, but their use in the treatment of HBV infection is limited as HBV-targeting immune cells are exhausted and HBV has evolved multiple mechanisms to evade and suppress IFN signalling. Thus, to fully utilize IFN-mediated intracellular mechanisms to effectively eliminate HBV, instead of direct IFN administration, novel strategies to sustain IFN-mediated anti-cccDNA and antiviral mechanisms need to be developed. This review will consolidate what is known about how IFNs act to achieve its intracellular antiviral effects and highlight the critical interferon-stimulated gene targets and effector mechanisms with potent anti-cccDNA functions. These include cccDNA degradation by APOBECs and cccDNA silencing and transcription repression by epigenetic modifications. In addition, the mechanisms that HBV employs to disrupt IFN signalling will be discussed. Drugs that have been developed or are in the pipeline for components of the IFN signalling pathway and HBV targets that detract IFN signalling mechanisms will also be identified and discussed for utility in the treatment of HBV infections. Together, these will provide useful insights into design strategies that specifically target cccDNA for the eradication of HBV.
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Affiliation(s)
- Zhi Yi Goh
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore
- Integrative Sciences and Engineering Programme, NUS Graduate School, National University of Singapore, Singapore 119077, Singapore
| | - Ee Chee Ren
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119260, Singapore
| | - Hui Ling Ko
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore
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Zhen S, Qiang R, Lu J, Tuo X, Yang X, Li X. Enhanced antiviral benefit of combination therapy with anti-HBV and anti-PD1 gRNA/cas9 produces a synergistic antiviral effect in HBV infection. Mol Immunol 2021; 130:7-13. [PMID: 33340931 DOI: 10.1016/j.molimm.2020.12.004] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2020] [Revised: 11/16/2020] [Accepted: 12/02/2020] [Indexed: 02/08/2023]
Abstract
Targeted therapy for patients with hepatitis B virus (HBV) infection can lead to objective responses, although response times may be short. At the same time, the response rate to programmed cell death-1 (PD-1) treatment was more durable. It is speculated that HBV targeted therapy can synergistically enhance the antitumor activity with PD-1 blockade. To test this hypothesis, we evaluated the effect of crispr-cas9 on HBV and PD-1 in vitro and in vivo. We found that HBV targeting gRNA/cas9 induced a decrease in the expression of HBsAg, while the PD-1 gene could be knocked out by electroporation targeting gRNA / cas9 by polymerase chain reaction. In HBV transgenic mice, the immunophenotype and cytokine expression of human dendritic cells (DCS) were detected by crispr-cas9 system stimulation, flow cytometry and polymerase chain reaction. These results indicate that gRNA/cas9 treatment upregulates the expression of CD80, CD83 and CD86, and significantly increases the mRNA levels of IL-6, IL-12, IL-23 and tumor necrosis factor alpha. The combination of anti HBV and anti PD-1 therapy can inhibit HBV expression and significantly improve the survival of HBV transgenic mice. In addition, the combination therapy increased the production of interferon by T cells, and then enhanced the expression of Th1 related immunostimulatory genes, thereby reducing the transcription of regulatory / inhibitory immune genes. In general, this response can reshape the tumor microenvironment from immunosuppression to immune stimulation. Finally, anti HBV therapy can induce the expression of interferon dependent programmed cell death ligand-1 in HBV transgenic mice in vivo. To sum up, these results demonstrate that the combination of HBV targeted therapy and PD-1 immune checkpoint block has a strong synergistic effect, thus supporting the transformation potential of this combined therapy strategy in clinical treatment of HBV infection.
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Affiliation(s)
- Shuai Zhen
- Medical Heredity Research Center, Northwest Women's and Children's Hospital, Shaanxi, PR China.
| | - Rong Qiang
- Medical Heredity Research Center, Northwest Women's and Children's Hospital, Shaanxi, PR China
| | - Jiaojiao Lu
- Center for Translational Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, PR China
| | - Xiaoqian Tuo
- Center for Translational Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, PR China
| | - Xiling Yang
- Center for Translational Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, PR China
| | - Xu Li
- Center for Translational Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, PR China
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Abstract
Antiviral drugs have traditionally been developed by directly targeting essential viral components. However, this strategy often fails due to the rapid generation of drug-resistant viruses. Recent genome-wide approaches, such as those employing small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeats (CRISPR) or those using small molecule chemical inhibitors targeting the cellular "kinome," have been used successfully to identify cellular factors that can support virus replication. Since some of these cellular factors are critical for virus replication, but are dispensable for the host, they can serve as novel targets for antiviral drug development. In addition, potentiation of immune responses, regulation of cytokine storms, and modulation of epigenetic changes upon virus infections are also feasible approaches to control infections. Because it is less likely that viruses will mutate to replace missing cellular functions, the chance of generating drug-resistant mutants with host-targeted inhibitor approaches is minimized. However, drug resistance against some host-directed agents can, in fact, occur under certain circumstances, such as long-term selection pressure of a host-directed antiviral agent that can allow the virus the opportunity to adapt to use an alternate host factor or to alter its affinity toward the target that confers resistance. This review describes novel approaches for antiviral drug development with a focus on host-directed therapies and the potential mechanisms that may account for the acquisition of antiviral drug resistance against host-directed agents.
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Novel Hepatitis B Virus Capsid Assembly Modulator Induces Potent Antiviral Responses In Vitro and in Humanized Mice. Antimicrob Agents Chemother 2020; 64:AAC.01701-19. [PMID: 31712213 DOI: 10.1128/aac.01701-19] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2019] [Accepted: 10/23/2019] [Indexed: 12/12/2022] Open
Abstract
Hepatitis B virus (HBV) affects an estimated 250 million chronic carriers worldwide. Though several vaccines exist, they are ineffective for those already infected. HBV persists due to the formation of covalently closed circular DNA (cccDNA)-the viral minichromosome-in the nucleus of hepatocytes. Current nucleoside analogs and interferon therapies rarely clear cccDNA, requiring lifelong treatment. Our group identified GLP-26, a novel glyoxamide derivative that alters HBV nucleocapsid assembly and prevents viral DNA replication. GLP-26 exhibited single-digit nanomolar anti-HBV activity, inhibition of HBV e antigen (HBeAg) secretion, and reduced cccDNA amplification, in addition to showing a promising preclinical profile. Strikingly, long term combination treatment with entecavir in a humanized mouse model induced a decrease in viral loads and viral antigens that was sustained for up to 12 weeks after treatment cessation.
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Exonuclease I and III improve the detection efficacy of hepatitis B virus covalently closed circular DNA. Hepatobiliary Pancreat Dis Int 2019; 18:458-463. [PMID: 30522829 DOI: 10.1016/j.hbpd.2018.11.003] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/14/2018] [Accepted: 10/29/2018] [Indexed: 02/05/2023]
Abstract
BACKGROUND Hepatitis B virus covalently closed circular DNA (HBV cccDNA) is an important biomarker of hepatitis B virus infection. However, the current methods are not specific and sensitive. The present study aimed to develop a specific and sensitive assay method for the quantification of HBV cccDNA. METHODS Exonuclease I (Exo I) & Exonuclease III (Exo III) and specific primer probes are used in real-time PCR. The virus particles isolated from peripheral blood mononuclear cells were used as negative control and HBV1.3 recombinant plasmid 3.2 kb circular DNA fragment was used as positive control. The methods of cccDNA detection were evaluated in cell lines, plasmid, animal model, patient serum and liver biopsies. RESULTS A linear range of 101-107 copies/assay using specific primers for HBV cccDNA was established. HBV cccDNA were only detected in cell lines, animal model and liver tissue. It cannot be detected in serum samples. Intrahepatic HBV cccDNA level had good correlation with intrahepatic total HBV DNA level (r = 0.765, P < 0.001). CONCLUSIONS The real-time quantitative PCR is an effective and feasible method for sensitive and specific detection of low copy number of cccDNA. The novel detection method is fast, provides high sensitivity and specificity and can be used in clinical practice.
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Kostyusheva A, Kostyushev D, Brezgin S, Volchkova E, Chulanov V. Clinical Implications of Hepatitis B Virus RNA and Covalently Closed Circular DNA in Monitoring Patients with Chronic Hepatitis B Today with a Gaze into the Future: The Field Is Unprepared for a Sterilizing Cure. Genes (Basel) 2018; 9:E483. [PMID: 30301171 PMCID: PMC6210151 DOI: 10.3390/genes9100483] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2018] [Revised: 09/30/2018] [Accepted: 10/03/2018] [Indexed: 12/12/2022] Open
Abstract
. Chronic hepatitis B virus (HBV) infection has long remained a critical global health issue. Covalently closed circular DNA (cccDNA) is a persistent form of the HBV genome that maintains HBV chronicity. Decades of extensive research resulted in the two therapeutic options currently available: nucleot(s)ide analogs and interferon (IFN) therapy. A plethora of reliable markers to monitor HBV patients has been established, including the recently discovered encapsidated pregenomic RNA in serum, which can be used to determine treatment end-points and to predict the susceptibility of patients to IFN. Additionally, HBV RNA splice variants and cccDNA and its epigenetic modifications are associated with the clinical course and risks of hepatocellular carcinoma (HCC) and liver fibrosis. However, new antivirals, including CRISPR/Cas9, APOBEC-mediated degradation of cccDNA, and T-cell therapies aim at completely eliminating HBV, and it is clear that the diagnostic arsenal for defining the long-awaited sterilizing cure is missing. In this review, we discuss the currently available tools for detecting and measuring HBV RNAs and cccDNA, as well as the state-of-the-art in clinical implications of these markers, and debate needs and goals within the context of the sterilizing cure that is soon to come.
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Affiliation(s)
| | | | - Sergey Brezgin
- Central Research Institute of Epidemiology, Moscow, 111123, Russia.
- National Research Centre, Institute of Immunology, Federal Medical Biological Agency, Moscow, 115478, Russia.
| | - Elena Volchkova
- I.M. Sechenov First Moscow State Medical University, Ministry of Health of the Russian Federation, Moscow, 119146, Russia.
| | - Vladimir Chulanov
- Central Research Institute of Epidemiology, Moscow, 111123, Russia.
- I.M. Sechenov First Moscow State Medical University, Ministry of Health of the Russian Federation, Moscow, 119146, Russia.
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Influential Factors of Hepatitis B Virus cccDNA in Peripheral Blood Mononuclear Cells Among HBsAg-Positive Pregnant Females Neonates. HEPATITIS MONTHLY 2018. [DOI: 10.5812/hepatmon.55064] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
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Kostyushev DS, Zueva AP, Brezgin SA, Lipatnikov AD, Simirskii VN, Glebe D, Volchkova EV, Shipulin GA, Chulanov VP. [Overexpression of DNA-methyltransferases in persistency of cccDNA pool in chronic hepatitis B]. TERAPEVT ARKH 2018; 89:21-26. [PMID: 29260742 DOI: 10.17116/terarkh2017891121-26] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
AIM To define the role of DNA-methyltransferases of type 1 and type 3A in hepatitis B viral cycle. MATERIAL AND METHODS Human hepatoma cells HepG2 with stable expression of 1.1-mer HBV genome were transfected with vectors encoding DNA-methyltransferase 1 (DNMT1), DNA-methyltransferase 3A (DNMT3A) or were co-transfected with these vectors. Total HBV DNA copy number, relative expression of pregenomic RNA (pgRNA), S-protein-encoding RNA (S-RNA) and cccDNA were analyzed by quantitative and semi-quantitative real-time PCR-analysis with TaqMan probes for assessment of DNMTs-mediated effects on HBV. RESULTS DNMT1 and DNMT3A suppress HBV transcription and replication, though to different magnitude. cccDNA pool is enlarged statistically significantly ≈2-fold (P<0.005) after transfection of DNMT3A, but is unaltered under DNMT1 treatment. CONCLUSION DNMT3A regulates the size of cccDNA pool and is important for persistency of HBV infection.
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Affiliation(s)
- D S Kostyushev
- Central Research Institute of Epidemiology, Moscow, Russia
| | - A P Zueva
- Central Research Institute of Epidemiology, Moscow, Russia; M.V. Lomonosov Moscow State University, Moscow, Russia
| | - S A Brezgin
- Central Research Institute of Epidemiology, Moscow, Russia; I.M. Sechenov First State Medical University, Moscow, Russia
| | - A D Lipatnikov
- Central Research Institute of Epidemiology, Moscow, Russia; D.I. Mendeleev University of Chemical Technology of Russia, Moscow, Russia
| | - V N Simirskii
- N.K. Koltzov Institute of Developmental Biology, Moscow, Russia
| | - D Glebe
- Justus-Liebig University of Giessen, Institute of Medical Virology, Giessen, Germany
| | - E V Volchkova
- I.M. Sechenov First State Medical University, Moscow, Russia
| | - G A Shipulin
- Central Research Institute of Epidemiology, Moscow, Russia
| | - V P Chulanov
- Central Research Institute of Epidemiology, Moscow, Russia; I.M. Sechenov First State Medical University, Moscow, Russia
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15
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Tsai HW, Lin YJ, Wu HC, Chang TT, Wu IC, Cheng PN, Yen CJ, Chan SH, Huang W, Su IJ. Resistance of ground glass hepatocytes to oral antivirals in chronic hepatitis B patients and implication for the development of hepatocellular carcinoma. Oncotarget 2017; 7:27724-34. [PMID: 27027237 PMCID: PMC5053683 DOI: 10.18632/oncotarget.8388] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2015] [Accepted: 03/08/2016] [Indexed: 12/13/2022] Open
Abstract
Ground glass hepatocytes (GGHs) have been shown to predict the development of hepatocellular carcinoma (HCC). Type I GGH and type II GGH harbor hepatitis B virus (HBV) pre-S1 and pre-S2 deletion mutants, respectively. Whether anti-HBV therapy can inhibit the expression of GGHs and potentially reduce HCC development is explored in this study. Two sets of liver specimens were included: the first contained 31 paired biopsy specimens obtained from chronic HBV patients receiving oral nucleos(t)ide analogue (NA) treatment; the second contained 186 resected liver tissues obtained from HBV-related HCC patients receiving surgery: 82 received NA before surgery and 104 did not. Compared with the baseline biopsy specimens, type I (P=0.527) and type II GGH (P=0.077) were not significantly decreased after 48 weeks of NA treatment in the first set of patients. In the second set, despite suppression of viral load (P<0.001) and periportal necrosis (P=0.006) in treated patients, GGH (P=0.594), cccDNA (P=0.172) and serum pre-S mutants (p=0.401) were not significantly suppressed. A significant decrease of type I (P=0.049) and type II GGH (P=0.029) could only be observed in patients after long duration of treatment (median duration: 4.3 years). In the treated patients, the persisted type II GGH remained an independent variable associated with decreased local recurrence-free survival of HCC (P=0.019) as in non-treated patients (P=0.001). In conclusion, the persistence of GGHs could explain the residual risk of HCC development under anti-HBV treatment. Therefore, intrahepatic GGHs and pre-S mutant are potential additional targets for HCC prevention in patients already receiving anti-HBV treatment.
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Affiliation(s)
- Hung-Wen Tsai
- Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan.,Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.,Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan
| | - Yih-Jyh Lin
- Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Han-Chieh Wu
- National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan
| | - Ting-Tsung Chang
- Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan.,Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - I-Chin Wu
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Pin-Nan Cheng
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Chia-Jui Yen
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Shih-Huang Chan
- Department of Statistics, National Cheng Kung University, Tainan, Taiwan
| | - Wenya Huang
- Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan.,Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Ih-Jen Su
- Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan.,National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan.,Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan
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16
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Liu Z, Jiang L, Liang G, Song E, Jiang W, Zheng Y, Gong C. Hepatitis B virus reactivation in breast cancer patients undergoing chemotherapy: A review and meta-analysis of prophylaxis management. J Viral Hepat 2017; 24:561-572. [PMID: 28072494 DOI: 10.1111/jvh.12672] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Accepted: 12/15/2016] [Indexed: 12/15/2022]
Abstract
Hepatitis B virus (HBV) reactivation during or after chemotherapy in patients with breast cancer has become a remarkable clinical problem. Prophylactic nucleos(t)ide analogues (NAs) are recommended for patients with breast cancer who are hepatitis B surface antigen (HBsAg) positive before chemotherapy. We performed an up-to-date meta-analysis to compare the efficacy of prophylactic lamivudine use with nonprophylaxis in HBsAg-positive breast cancer patients undergoing chemotherapy. PubMed, the Cochrane Library and China National Knowledge Infrastructure (CNKI) databases were searched for relevant articles until June 2016. Eligible articles comparing the efficacy of prophylactic lamivudine use with nonprophylaxis in HBsAg-positive breast cancer patients undergoing chemotherapy were identified. Eight studies which had enrolled 709 HBsAg-positive breast cancer patients undergoing chemotherapy were analysed. Lamivudine prophylaxis significantly reduced the rates of chemotherapy-associated hepatitis B flares in chronic hepatitis B in breast cancer compared with patients with nonprophylaxis (odds ratio [OR]=0.15, 95% confidence interval [CI]: 0.07-0.35, P<.00001). Chemotherapy disruption rates attributed to HBV reactivation in the prophylaxis groups were significantly lower than the nonprophylaxis groups (OR=0.17, 95% CI: 0.07-0.43, P=.0002). Patients with lamivudine prophylaxis had a higher risk for tyrosine-methionine-aspartate-aspartate (YMDD) motif mutations than patients with nonprophylaxis (OR=6.33, 95% CI: 1.01-39.60, P=.05). Prophylactic antiviral therapy management is necessary for HBsAg-positive breast cancer patients undergoing chemotherapy, in spite of high correlation with lamivudine-resistant HBV variants with YMDD motif mutations.
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Affiliation(s)
- Z Liu
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetic and Gene Regulation, Breast Tumor Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - L Jiang
- Department of oncology Surgery, The First Hospital of Lanzhou University, Lanzhou, China
| | - G Liang
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetic and Gene Regulation, Breast Tumor Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - E Song
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetic and Gene Regulation, Breast Tumor Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - W Jiang
- Cardiff China Medical Research Collaborative, School of Medicine, Cardiff University, Cardiff, UK
| | - Y Zheng
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.,Division of Medicine, Liver Failure Group ILDH, UCL Medical School, London, UK
| | - C Gong
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetic and Gene Regulation, Breast Tumor Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.,Cardiff China Medical Research Collaborative, School of Medicine, Cardiff University, Cardiff, UK
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17
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Li X, Zhao J, Yuan Q, Xia N. Detection of HBV Covalently Closed Circular DNA. Viruses 2017; 9:E139. [PMID: 28587292 PMCID: PMC5490816 DOI: 10.3390/v9060139] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2017] [Revised: 05/30/2017] [Accepted: 05/30/2017] [Indexed: 12/19/2022] Open
Abstract
Chronic hepatitis B virus (HBV) infection affects approximately 240 million people worldwide and remains a serious public health concern because its complete cure is impossible with current treatments. Covalently closed circular DNA (cccDNA) in the nucleus of infected cells cannot be eliminated by present therapeutics and may result in persistence and relapse. Drug development targeting cccDNA formation and maintenance is hindered by the lack of efficient cccDNA models and reliable cccDNA detection methods. Southern blotting is regarded as the gold standard for quantitative cccDNA detection, but it is complicated and not suitable for high-throughput drug screening, so more sensitive and simple methods, including polymerase chain reaction (PCR)-based methods, Invader assays, in situ hybridization and surrogates, have been developed for cccDNA detection. However, most methods are not reliable enough, and there are no unified standards for these approaches. This review will summarize available methods for cccDNA detection. It is hoped that more robust methods for cccDNA monitoring will be developed and that standard operation procedures for routine cccDNA detection in scientific research and clinical monitoring will be established.
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Affiliation(s)
- Xiaoling Li
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China.
| | - Jinghua Zhao
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen 361102, China.
| | - Quan Yuan
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China.
| | - Ningshao Xia
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China.
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen 361102, China.
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18
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Sun YH, Lei XY, Sai YP, Chen JH, Sun YC, Gao X. Relationship between genotypes and clinical manifestation, pathology, and cccDNA in Chinese children with hepatitis B virus-associated glomerulonephritis. World J Pediatr 2016; 12:347-352. [PMID: 27059747 DOI: 10.1007/s12519-016-0015-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/29/2014] [Accepted: 12/11/2014] [Indexed: 12/30/2022]
Abstract
BACKGROUND Hepatitis B virus-associated glomerulonephritis (HBV-GN) is one of the extrahepatic manifestations after HBV infection, which would cause great clinical harm to people. The present study was undertaken to investigate the HBV-GN genotypes and its clinical relevance in Chinese children. METHODS A total of 41 HBV-infected children diagnosed with HBV-GN were enrolled in the study. All patients underwent liver and kidney biopsy. The genotypes and cccDNA were detected in their serum samples to analyze the relationship between HBV genotypes and clinical characteristics, cccDNA, and pathology. RESULTS Among the 41 children with HBV-GN, 29 (70.7%) had genotype C, 10 (24.4%) had genotype B, 2 (4.9%) had genotype B/C, and none of them had genotype non-B/C. Most children had genotypes B or C; moreover, the genotype C was the most frequent one. The incidence of hematuria and albuminuria, reduction in complement C3, increase in serum alanine aminotransferase levels and renal insufficiency in the children with genotype C were significantly higher than those in the children with genotype B (P<0.05); however, there was no statistically significant difference in hypertension and hepatomegaly (P>0.05). The frequency of HBV cccDNA positive in the genotype C group was significantly higher than that in the genotype B group (72.4% vs. 30.0%, P<0.05). No difference was observed in hepatic inflammation grades and stages of fibrosis between the two groups (P>0.05). CONCLUSIONS Genotype C was the most frequent genotype in the described group of patients with HBV-GN, and the liver and kidney damage indicators were more likely to occur in patients with genotype C.
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Affiliation(s)
- Yong-Hong Sun
- Department of Pediatrics, People's Hospital of Gansu Province, Lanzhou, Gansu, China
| | - Xiao-Yan Lei
- Department of Pediatrics, People's Hospital of Gansu Province, Lanzhou, Gansu, China.
| | - Yi-Pa Sai
- Department of Pediatrics, People's Hospital of Gansu Province, Lanzhou, Gansu, China
| | - Jun-Hui Chen
- Department of Pediatrics, People's Hospital of Gansu Province, Lanzhou, Gansu, China
| | - Yuan-Chun Sun
- Department of Pediatrics, People's Hospital of Gansu Province, Lanzhou, Gansu, China
| | - Xia Gao
- Department of Pediatrics, People's Hospital of Gansu Province, Lanzhou, Gansu, China
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19
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Tajik Z, Keyvani H, Bokharaei-Salim F, Zolfaghari MR, Fakhim S, Keshvari M, Alavian SM. Detection of Hepatitis B Virus Covalently Closed Circular DNA in the Plasma of Iranian HBeAg-Negative Patients With Chronic Hepatitis B. HEPATITIS MONTHLY 2015; 15:e30790. [PMID: 26504471 PMCID: PMC4612772 DOI: 10.5812/hepatmon.30790] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/15/2015] [Revised: 08/16/2015] [Accepted: 08/30/2015] [Indexed: 12/11/2022]
Abstract
BACKGROUND Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a marker of HBV replication in the liver of patients infected with HBV. OBJECTIVES This study aimed to investigate the association between the presence of cccDNA in the plasma samples of Iranian treatment-naive patients with chronic hepatitis B infection and HBV viral load and HBsAg levels. PATIENTS AND METHODS From April 2012 to May 2015, 106 treatment-naive patients with chronic hepatitis B infection were enrolled in this cross-sectional study. The HBsAg titer was measured by the Roche HBsAg II assay on the Cobas e411 system, and HBV DNA quantitation was performed using the COBAS TaqMan 48 kit. Real-time polymerase chain reaction was performed for the detection of HBV cccDNA. RESULTS The mean (SD) age of the patients was 41.1 ± 12.4 years (range, 20 - 62 years). From a total of 106 study participants, 67 (63.2%) were males. The HBV cccDNA was detected in plasma specimens in 19 (17.9%) out of the total 106 patients, and a significant relationship was found between the presence of cccDNA in plasma sample of males (23.9%) and females (7.7%) (P = 0.039). Also, a significant correlation was found between the presence of cccDNA in plasma sample of the patients and HBV viral load level (P < 0.0001) and HBsAg titer (P = 0.0043). CONCLUSIONS This study showed that cccDNA can be detected in the plasma specimen of 17.9% of Iranian treatment-naive patients with chronic hepatitis B infection. Therefore, designing prospective studies focusing on the detection of cccDNA in these patients would provide more information.
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Affiliation(s)
- Zahra Tajik
- Department of Microbiology, Faculty of Basic Sciences, Qom Branch, Islamic Azad University, Qom, IR Iran
| | - Hossein Keyvani
- Department of Virology, Iran University of Medical Sciences, Tehran, IR Iran
| | - Farah Bokharaei-Salim
- Department of Virology, Iran University of Medical Sciences, Tehran, IR Iran
- HIV Laboratory of National Center, Deputy of Health, Iran University of Medical Sciences, Tehran, IR Iran
- Corresponding Author: Farah Bokharaei-Salim, Department of Virology, Iran University of Medical Sciences, Tehran, IR Iran. Tel/Fax: +98-2188602205, E-mail:
| | - Mohammad Reza Zolfaghari
- Department of Microbiology, Faculty of Basic Sciences, Qom Branch, Islamic Azad University, Qom, IR Iran
| | - Shahin Fakhim
- Department of Civil Engineering, Faculty of Engineering, Payame Noor University, Karaj, IR Iran
| | - Maryam Keshvari
- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, IR Iran
| | - Seyed Moayed Alavian
- Middle East Liver Disease Center, Tehran, IR Iran
- Iran Hepatitis Network, Tehran, IR Iran
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20
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CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X. Sci Rep 2015; 5:13734. [PMID: 26334116 PMCID: PMC4558539 DOI: 10.1038/srep13734] [Citation(s) in RCA: 98] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2015] [Accepted: 08/04/2015] [Indexed: 02/07/2023] Open
Abstract
Current antiviral therapies cannot cure hepatitis B virus (HBV) infection; successful HBV eradication would require inactivation of the viral genome, which primarily persists in host cells as episomal covalently closed circular DNA (cccDNA) and, to a lesser extent, as chromosomally integrated sequences. However, novel designer enzymes, such as the CRISPR/Cas9 RNA-guided nuclease system, provide technologies for developing advanced therapy strategies that could directly attack the HBV genome. For therapeutic application in humans, such designer nucleases should recognize various HBV genotypes and cause minimal off-target effects. Here, we identified cross-genotype conserved HBV sequences in the S and X region of the HBV genome that were targeted for specific and effective cleavage by a Cas9 nickase. This approach disrupted not only episomal cccDNA and chromosomally integrated HBV target sites in reporter cell lines, but also HBV replication in chronically and de novo infected hepatoma cell lines. Our data demonstrate the feasibility of using the CRISPR/Cas9 nickase system for novel therapy strategies aiming to cure HBV infection.
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21
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Pal A, Sarkar N, Saha D, Guha SK, Saha B, Chakrabarti S, Chakravarty R. High incidence of lamivudine-resistance-associated vaccine-escape HBV mutants among HIV-coinfected patients on prolonged antiretroviral therapy. Antivir Ther 2015; 20:545-54. [PMID: 25654813 DOI: 10.3851/imp2942] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/29/2015] [Indexed: 02/04/2023]
Abstract
BACKGROUND Worldwide, frequent emergence of lamivudine (3TC)-resistant HBV mutants has been reported in HIV-HBV-coinfected patients during long-term antiretroviral therapy (ART) that contains 3TC as the sole anti-HBV drug. Three major patterns of mutations in HBV polymerase gene, namely single (rtM204V), double (rtL180M+rtM204V) and triple (rtV173L+rtL180M+rtM204V) mutations, are associated with 3TC-resistance; additionally, the triple mutation has vaccine-escape potential due to a corresponding change in overlapping surface gene. Data from India, a major reservoir for HIV and HBV infection, is lacking. Here we investigated the effect of long-term 3TC treatment on virological response for HBV and characterized the 3TC-resistant HBV mutations in a cohort of HIV-HBV-coinfected patients from eastern India. METHODS A cross-sectional study was performed in HIV-infected patients (n=563) receiving 3TC-containing ART for ≥6 months from the major ART centre of eastern India during 2011-2012. The hepatitis B surface antigen-positive HIV-infected patients (n=62) were categorized into four groups with comparable sample size according to the 3TC exposure for ≥6-<12 months (group I; n=15), ≥12-<24 months (group II; n=20), ≥24-<48 months (group III; n=13) and ≥48 months (group IV; n=14). Patients' plasma samples were examined for hepatitis B e antigen (HBeAg), HBV DNA, viral load and covalently closed circular DNA (cccDNA). HBV reverse transcriptase region was sequenced. RESULTS With a longer period of 3TC exposure, the frequency of HIV-HBV-coinfected patients having HBV DNA suppression decreased. The prevalence of HBeAg-positivity, serum HBV DNA load >2,000 IU/ml and 3TC-resistant mutations simultaneously increased. Remarkably, the 3TC-resistant triple mutation predominated over the double mutation in this cohort (32.26% versus 19.34%) and prevailed in significantly higher frequency among HBV viraemic patients experiencing 3TC for ≥48 months (60% versus 10%; P=0.03). Patients with 3TC-resistant triple mutants had HBV genotype-D, high serum HBV DNA load and elevated alanine aminotransferase level, and presence of cccDNA in their serum. CONCLUSIONS Considering this alarmingly high incidence of 3TC-resistant triple mutation and its possible clinical/public health implications, proper management of 3TC-resistance among HIV-HBV-coinfected patients is an urgent necessity in India.
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Affiliation(s)
- Ananya Pal
- ICMR Virus Unit, Kolkata, ID & BG Hospital Campus, Kolkata, India
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22
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Shi M, Sun WL, Hua YY, Han B, Shi L. Effects of entecavir on hepatitis B virus covalently closed circular DNA in hepatitis B e antigen-positive patients with hepatitis B. PLoS One 2015; 10:e0117741. [PMID: 25647607 PMCID: PMC4315599 DOI: 10.1371/journal.pone.0117741] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2014] [Accepted: 01/01/2015] [Indexed: 12/18/2022] Open
Abstract
The aim of this study was to assess the effect of 48-week entecavir therapy on serum and intrahepatic hepatitis B virus, covalently closed circular DNA (HBV cccDNA) levels in hepatitis B e antigen (HBeAg)-positive patients. A total of 120 patients with HBeAg-positive chronic hepatitis were treated with entecavir for 48 weeks. Serum HBV markers, total HBV DNA, and HBV cccDNA levels were measured at baseline and week 48. Biopsies from 20 patients were available for both intrahepatic total HBV DNA and cccDNA testing at these timepoints. HBV cccDNA levels were decreased from a median level of 5.1×106 copies/mL at baseline to a median level of 2.4×103 copies/mL at week 48. Reduction magnitudes of HBV cccDNA in patients with normalized alanine aminotransferase levels and those undergoing HBeAg seroconversion were significantly greater than those in alanine aminotransferase-abnormal and HBeAg positive patients. Intrahepatic HBV cccDNA was decreased significantly after 48 weeks of treatment, but could not be eradicated. In conclusion, treatment of HBeAg-positive hepatitis B patients with entecavir for 48 weeks decreased serum and intrahepatic HBV cccDNA significantly, and the magnitude of HBV cccDNA reduction was related to total HBV DNA decrease, alanine aminotransferase normalization, and HBeAg seroconversion.
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Affiliation(s)
- Ming Shi
- Department of Laboratory Medicine, No. 6 Hospital of Dalian, Dalian, China
- * E-mail:
| | - Wan-Li Sun
- Department of Laboratory Medicine, No. 6 Hospital of Dalian, Dalian, China
| | - Yan-Yan Hua
- Department of Laboratory Medicine, No. 6 Hospital of Dalian, Dalian, China
| | - Bo Han
- Department of Laboratory Medicine, No. 6 Hospital of Dalian, Dalian, China
| | - Long Shi
- Department of Laboratory Medicine, No. 6 Hospital of Dalian, Dalian, China
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23
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Guo Y, Sheng S, Nie B, Tu Z. Development of magnetic capture hybridization and quantitative polymerase chain reaction for hepatitis B virus covalently closed circular DNA. HEPATITIS MONTHLY 2015; 15:e23729. [PMID: 25741372 PMCID: PMC4344652 DOI: 10.5812/hepatmon.23729] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/22/2014] [Revised: 12/03/2014] [Accepted: 12/14/2014] [Indexed: 12/11/2022]
Abstract
BACKGROUND Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) served as a vital role in the life cycle of the virus and persistent infection. However, specific and quantitative methods for cccDNA detection have not been available. OBJECTIVES Our aim was to develop and primarily evaluate a quantitative method for HBV cccDNA based on magnetic capture hybridization and quantitative PCR technology. MATERIALS AND METHODS The functionalized-nanoparticles specifically to capture HBV cccDNA, located on both sides of relaxed circle DNA (rcDNA) gap, were designed. Then, magnetic capture hybridization and quantitative PCR (MCH-qPCR) assay were developed and its performance was primarily evaluated with cccDNA standards and serum samples of patients with chronic hepatitis B. RESULTS Specific nanoparticles of cccDNA capture were prepared and a magnetic capture hybridization and quantitative assay method for cccDNA was developed successfully. The limit of detection was 90 IU/mL, and a good linear relationship in the range of 10(2)-10(6) IU/mL was revealed (r(2) = 0.994) with the MCH-qPCR. Compared with directly real-time PCR, a high content of HBV DNA did not affect the detection of cccDNA for the MCH-qPCR method, and there was no cross-reactivity between cccDNA and rcDNA. CONCLUSIONS The novel MCH-qPCR method has good sensitivity and specificity. It could meet the requirement of clinical routine detection.
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Affiliation(s)
- Yongcan Guo
- The Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Shangchun Sheng
- The Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Bin Nie
- The Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Zhiguang Tu
- The Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, China
- Corresponding Author: Zhiguang Tu, The Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, China. Tel: +86-2368485759, Fax: +86-2368485239, E-mail:
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24
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Kennedy EM, Bassit LC, Mueller H, Kornepati AVR, Bogerd HP, Nie T, Chatterjee P, Javanbakht H, Schinazi RF, Cullen BR. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology 2014; 476:196-205. [PMID: 25553515 DOI: 10.1016/j.virol.2014.12.001] [Citation(s) in RCA: 183] [Impact Index Per Article: 16.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2014] [Revised: 11/14/2014] [Accepted: 12/01/2014] [Indexed: 02/06/2023]
Abstract
Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.
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Affiliation(s)
- Edward M Kennedy
- Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC, United States
| | - Leda C Bassit
- Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, United States
| | - Henrik Mueller
- Infectious Diseases, F. Hoffmann-LaRoche, Inc., Basel, Switzerland
| | - Anand V R Kornepati
- Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC, United States
| | - Hal P Bogerd
- Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC, United States
| | - Ting Nie
- Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, United States
| | - Payel Chatterjee
- Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, United States
| | | | - Raymond F Schinazi
- Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, United States.
| | - Bryan R Cullen
- Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC, United States.
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25
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Golsaz Shirazi F, Mohammadi H, Amiri MM, Singethan K, Xia Y, Bayat AA, Bahadori M, Rabbani H, Jeddi-Tehrani M, Protzer U, Shokri F. Monoclonal antibodies to various epitopes of hepatitis B surface antigen inhibit hepatitis B virus infection. J Gastroenterol Hepatol 2014; 29:1083-91. [PMID: 24325676 DOI: 10.1111/jgh.12483] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 11/13/2013] [Indexed: 12/29/2022]
Abstract
BACKGROUND AND AIM Antibodies against the "a" determinant of hepatitis B surface antigen (HBsAg) are able to neutralize circulating hepatitis B virus (HBV) particles and prevent HBV infection. It has been proposed that a single amino acid exchange may allow the virus to escape the immune response. We used a set of monoclonal antibodies (MAbs) to investigate whether a single mutation may account for virus escape from humoral immunity. METHODS Nine murine HBsAg-specific MAbs were raised. Reactivity of all antibodies with 14 recombinant mutants of HBsAg was assessed by ELISA. HBV infection of HepaRG cells was used to evaluate viral neutralization capacity of MAbs in vitro. RESULTS All MAbs were able to inhibit the establishment of HBV infection in a dose-dependent fashion, but recognition of HBsAg variants varied. The MAbs were classified into three subgroups based on their pattern of reactivity to the HBsAg variants. Accordingly, three MAbs showed weak reactivity (< 40%) to variants with mutations within the first loop of "a" determinant, five MAbs displayed negligible binding to variants with mutations within the second loop, and one MAb lost its binding to variants having mutations in both loops of the "a" determinant. CONCLUSIONS Our results indicate that antibodies against different epitopes of the "a" determinant of HBsAg are able to neutralize HBV. It seems that mutations within a single or a limited number of amino acids within this determinant can hardly result in viral escape. These results have important implications for the development of antibody-based therapies against HBV.
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Affiliation(s)
- Forough Golsaz Shirazi
- Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
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26
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Singla B, Chakraborti A, Sharma BK, Kapil S, Chawla YK, Arora SK, Das A, Dhiman RK, Duseja A. Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients. Mol Biol Rep 2014; 41:4689-96. [PMID: 24706057 DOI: 10.1007/s11033-014-3339-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2013] [Accepted: 03/21/2014] [Indexed: 02/07/2023]
Abstract
Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7%) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1% HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.
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Affiliation(s)
- Bhupesh Singla
- Department of Hepatology, Nehru Hospital, Post Graduate Institute of Medical Education and Research, Chandigarh, 160012, India
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27
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Martel N, Gomes SA, Chemin I, Trépo C, Kay A. Improved rolling circle amplification (RCA) of hepatitis B virus (HBV) relaxed-circular serum DNA (RC-DNA). J Virol Methods 2013; 193:653-659. [PMID: 23928222 DOI: 10.1016/j.jviromet.2013.07.045] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2013] [Revised: 07/22/2013] [Accepted: 07/26/2013] [Indexed: 12/14/2022]
Abstract
For functional analysis of HBV isolates, epidemiological studies and correct identification of recombinant genomes, the amplification of complete genomes is necessary. A method for completely in vitro amplification of full-length HBV genomes starting from serum RC-DNA is described. This uses in vitro completion/ligation of plus-strand HBV RC-DNA and amplification using Rolling-Circle Amplification, eventually followed by a genomic PCR. The method can amplify complete HBV genomes from sera with viral loads ranging from >1.0E+8 IU/ml down to 1.0E+3 IU/ml. The method can be applied to archived sera that have undergone long-term storage or to archived DNA serum extracts. The genomes can easily be cloned. HBV genotypes A-G can all be amplified with no apparent problems. A recombinant subgenotype A3/genotype E genome was identified and fully sequenced.
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Affiliation(s)
- Nora Martel
- Centre de Recherche en Cancérologie de Lyon (CRCL); INSERM, U1052; CNRS, UMR 5286; UCBL1, S_1052. 151 cours Albert Thomas, 69003 Lyon, France
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28
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Schildgen V, Malecki M, Tillmann RL, Brockmann M, Schildgen O. The Human Bocavirus Is Associated with Some Lung and Colorectal Cancers and Persists in Solid Tumors. PLoS One 2013; 8:e68020. [PMID: 23826357 PMCID: PMC3694905 DOI: 10.1371/journal.pone.0068020] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2013] [Accepted: 05/24/2013] [Indexed: 02/07/2023] Open
Abstract
Human bocavirus is the second autonomous human parvovirus with assumed pathogenic potential. Other parvoviruses are known to persist and even integrate into the host genome, eventually contributing to the multi-step development of cancer. Human bocavirus also persists in an unknown percentage of clinically asymptomatic patients in addition to those with primary infection. The aim of the present study was to analyze the role of Human bocavirus in lung and colorectal cancers. Therefore, formalin-fixed, paraffin-embedded, archived tumor samples were screened for Human bocavirus DNA by PCR, Southern blotting, and sequencing. Positive tissues were further subjected to fluorescence in situ hybridization analysis to specifically detect human bocavirus DNA in the infected cells. In total, 11 of the 60 (18.3%) lung and 9 of the 44 (20.5%) colorectal tumors tested positive for human bocavirus DNA by PCR and were confirmed by sequencing and fluorescence in situ hybridization analysis. Thus, human bocavirus DNA is present in the nuclei of infected cells, in either single or multiple copies, and appears to form concatemers. The occurrence of these human bocavirus DNA structures supports the existence of the postulated σ- or rolling-hairpin replication mechanism. Moreover, the fluorescence in situ hybridization patterns inspired the hypothesis that human bocavirus DNA either persists as cccDNA or is integrated into the host genome. This finding suggests that this virus may indirectly contribute to the development of some colorectal and lung cancers, as do other DNA viruses, such as the human hepatitis B virus, or may play an active role in cancer by interacting with the host genome.
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Affiliation(s)
- Verena Schildgen
- Kliniken der Stadt Köln gGmbH, Klinikum der Privaten Universität Witten-Herdecke mit Sitz in Köln, Institut für Pathologie, Köln, Germany
| | - Monika Malecki
- Kliniken der Stadt Köln gGmbH, Klinikum der Privaten Universität Witten-Herdecke mit Sitz in Köln, Institut für Pathologie, Köln, Germany
| | - Ramona-Liza Tillmann
- Kliniken der Stadt Köln gGmbH, Klinikum der Privaten Universität Witten-Herdecke mit Sitz in Köln, Institut für Pathologie, Köln, Germany
| | - Michael Brockmann
- Kliniken der Stadt Köln gGmbH, Klinikum der Privaten Universität Witten-Herdecke mit Sitz in Köln, Institut für Pathologie, Köln, Germany
| | - Oliver Schildgen
- Kliniken der Stadt Köln gGmbH, Klinikum der Privaten Universität Witten-Herdecke mit Sitz in Köln, Institut für Pathologie, Köln, Germany
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29
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Shen G, Fu X, Zhou B, Yin J, Zhong C, Chen J, Hou J. Duck HBV DNA copy numbers in isolated hepatocyte nuclei vary dramatically and decline during entecavir therapy. Antivir Ther 2013; 18:987-996. [PMID: 23765241 DOI: 10.3851/imp2653] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/12/2013] [Indexed: 10/26/2022]
Abstract
BACKGROUND We aimed to develop a quantitative assay to measure duck HBV (DHBV) DNA in single hepatocyte nuclei from DHBV-infected animals and to observe intranuclear DHBV DNA kinetics undergoing entecavir (ETV) therapy. METHODS DHBV DNA in isolated nuclei was amplified by quantitative real-time PCR. Liver tissues from chronically-infected ducks with or without ETV treatment were assessed. Cell cycle phases were defined with flow cytometry in single nuclei. RESULTS We successfully established a quantitative assay to measure intranuclear DHBV DNA in single nuclei with high specificity, sensitivity and acceptable interassay variations. The intranuclear viral DNA copy numbers varied dramatically (2-204 copies/nuclei) in 11 ducks with active viral replication. Average intranuclear DHBV DNA copies from individual animals (7.57-57.67 copies/nuclei) significantly correlated with total intranuclear (rs=0.955, P<0.001) and serum (rs=0.745, P=0.008) viral DNA levels. The median intranuclear DHBV DNA copies in virus-positive nuclei were greater in gap 0/1 than those in gap 2/mitosis and synthesis phases (P<0.001). Median intranuclear viral DNA copies in virus-positive nuclei decreased from 21 to 6 (P<0.001) under 14-19 weeks of ETV therapy. However, subsequently, further reductions were not achieved in four animals after extended 16 week treatment (6 versus 11, P=0.034). CONCLUSIONS Intranuclear DHBV DNA levels varied significantly, which could be partially attributed to effects of cell cycle phases, and could be decreased by ETV therapy.
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Affiliation(s)
- Guojun Shen
- Hepatology Unit and Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, PR China
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30
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Ma J, Huang C, Yao X, Shi C, Sun L, Yuan L, Lei P, Zhu H, Liu H, Wu X, Ning Q, Zhou C, Shen G. Inhibition of hepatitis B virus and induction of hepatoma cell apoptosis by ASGPR-directed delivery of shRNAs. PLoS One 2012; 7:e46096. [PMID: 23094023 PMCID: PMC3477153 DOI: 10.1371/journal.pone.0046096] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2012] [Accepted: 08/28/2012] [Indexed: 01/23/2023] Open
Abstract
Hepatitis B virus (HBV) infection is a worldwide liver disease and nearly 25% of chronic HBV infections terminate in hepatocellular carcinoma (HCC). Currently, there is no effective therapy to inhibit HBV replication and to eliminate hepatoma cells, making it highly desired to develop novel therapies for these two stages of the HBV-caused detrimental disease. Recently, short hairpin RNA (shRNA) has emerged as a potential therapy for virus-infected disease and cancer. Here, we have generated a shRNA, pGenesil-siHBV4, which effectively inhibits HBV replication in the human hepatoma cell line HepG2.2.15. The inhibitory effects of pGenesil-siHBV4 are manifested by the decrease of both the HBV mRNA level and the protein levels of the secreted HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), and by the reduction of secreted HBV DNA. Using mouse hydrodynamic tail vein injection, we demonstrate that pGenesil-siHBV4 is effective in inhibiting HBV replication in vivo. Because survivin plays a key role in cancer cell escape from apoptosis, we further generated pGenesil-siSurvivin, a survivin-silencing shRNA, and showed its effect of triggering apoptosis of HBV-containing hepatoma cells. To develop targeted shRNA therapy, we have identified that as a specific binder of the asialoglycoprotein receptor (ASGPR), jetPEI-Hepatocyte delivers pGenesil-siHBV4 and pGenesil-siSurvivin specifically to hepatocytes, not other types of cells. Finally, co-transfection of pGenesil-siHBV4 and pGenesil-siSurvivin exerts synergistic effects in inducing hepatoma cell apoptosis, a novel approach to eliminate hepatoma by downregulating survivin via multiple mechanisms. The application of these novel shRNAs with the jetPEI-Hepatocyte targeting strategy demonstrates the proof-of-principle for a promising approach to inhibit HBV replication and eliminate hepatoma cells with high specificity.
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MESH Headings
- Animals
- Apoptosis
- Asialoglycoprotein Receptor/genetics
- Asialoglycoprotein Receptor/metabolism
- Carcinoma, Hepatocellular/etiology
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/therapy
- DNA, Viral/antagonists & inhibitors
- DNA, Viral/biosynthesis
- Gene Expression Regulation, Neoplastic
- Genetic Vectors
- Hepatitis B Surface Antigens/genetics
- Hepatitis B e Antigens/genetics
- Hepatitis B virus/genetics
- Hepatitis B virus/growth & development
- Hepatitis B, Chronic/complications
- Hepatitis B, Chronic/genetics
- Hepatitis B, Chronic/therapy
- Humans
- Inhibitor of Apoptosis Proteins/antagonists & inhibitors
- Inhibitor of Apoptosis Proteins/genetics
- Inhibitor of Apoptosis Proteins/metabolism
- Injections, Intravenous
- Liver/pathology
- Liver/virology
- Male
- Mice
- Mice, Inbred BALB C
- Molecular Targeted Therapy
- Organ Specificity
- RNA, Small Interfering/genetics
- RNA, Small Interfering/therapeutic use
- Repressor Proteins/antagonists & inhibitors
- Repressor Proteins/genetics
- Repressor Proteins/metabolism
- Survivin
- Transfection
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Affiliation(s)
- Jingwei Ma
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Chunmei Huang
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Xinxin Yao
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Chuan Shi
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Lifang Sun
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Lu Yuan
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Ping Lei
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Huifen Zhu
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Hongbo Liu
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Xiongwen Wu
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Qin Ning
- Department of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Chun Zhou
- Department of Environmental Health Sciences, Columbia University, New York, New York, United States of America
- * E-mail: (CZ); (GS)
| | - Guanxin Shen
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
- * E-mail: (CZ); (GS)
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31
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Inhibition on hepatitis B virus in vitro of lectin from Musca domestica pupa via the activation of NF-κB. Virus Res 2012; 170:53-8. [PMID: 22940568 DOI: 10.1016/j.virusres.2012.08.012] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2012] [Revised: 08/10/2012] [Accepted: 08/16/2012] [Indexed: 11/21/2022]
Abstract
The present study reported that the secretions of HBsAg and HBeAg in HepG2.2.15 cells were significantly decreased under the treatment of lectin from Musca domestica pupa (MPL). Both the replication of hepatitis B virus (HBV) DNA and HBV cccDNA in cells, and the copies of extracellular HBV DNA were inhibited by MPL. The mRNA expressions of interleukin-2 (IL-2), gamma interferon (INF-γ) and MxA were up-regulated by MPL treatments, but down-regulated when nuclear factor-κB (NF-κB) signal pathway was blocked by pyrrolidine dithiocarbamate (PDTC). Subsequent investigation revealed that nuclear factor-κB inhibitory κB (IκB) in endochylema was inhibited and NF-κB was translocated into the nucleus. These findings indicate that MPL could inhibit HBV replication via the induction of the expression of IL-2, INF-γ and MxA through the activation of NF-κB.
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32
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Relationship between HBV cccDNA expression in the human ovary and vertical transmission of HBV. Epidemiol Infect 2011; 140:1454-60. [PMID: 22000033 DOI: 10.1017/s0950268811002068] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
This study aimed to investigate the relationship between hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the ovary and vertical transmission of HBV. HBV DNA and HBV cccDNA were assayed in the ovaries of 33 pregnant women who were positive for HBV DNA. The HBVM (HBV markers, including HBsAg, HBsAb, HBeAg, HBeAb, HBcAb) level and the HBV DNA content in peripheral blood of infants were measured. The overall positive rate of HBV DNA and HBV cccDNA in samples was 51·52% (17/33). The intrauterine infection rate of the infants was 12·12% (4/33). When HBV DNA and HBV cccDNA were both positive, the intrauterine infection rate of infants was significantly higher than when they were both negative (P<0·05). Levels of HBV cccDNA and the rate of positive samples were significantly higher in mothers with infants with intrauterine infection than in those without (P<0·01 and P<0·05, respectively). HBV can infect the human ovary and may transmit to the filial generation via the ovum.
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Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR. Clin Chim Acta 2011; 412:1905-11. [PMID: 21741960 DOI: 10.1016/j.cca.2011.06.031] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2011] [Revised: 06/15/2011] [Accepted: 06/23/2011] [Indexed: 02/07/2023]
Abstract
BACKGROUND The study aimed to develop an effective method to quantitate HBV covalently closed circular DNA (cccDNA) using small section of formalin fixed paraffin-embedded (FFPE) liver biopsy. METHODS Plasmid-safe ATP-dependent DNase (PSAD)-treated samples were subjected to rolling circle amplification (RCA) prior to real-time PCR mediated by cccDNA-selective primers. Human beta-actin gene was used as a reference control. RESULTS Compared to the classical method, i.e., PSAD digestion+real-time PCR, introduction of RCA increased the lower limit of detection for about 2 logs with good inter- and intra-assay reproducibility. HBV cccDNA was detected in 91.5% (119/130) of the FFPE samples. The cccDNA levels (copy/cell) between FFPE liver tissues and fresh frozen counterpart tissues were comparable. The median of cccDNA level in HBeAg-positive patients was higher than that in HBeAg-negative ones (52.60 vs. 31.25copies/cell, P<0.01). Intrahepatic cccDNA level was positively correlated with intrahepatic HBV total DNA level, but not obviously correlated with serum HBV DNA or alanine aminotransferase levels. CONCLUSIONS The method could sensitively and specifically quantitate intrahepatic HBV cccDNA in micro FFPE liver biopsy tissue for evaluation of HBV replication status in the liver.
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Ding Y, Ma L, Wang XZ, Zhang J, Zhao GZ, Wang ZQ, Dou XG. In vitro study on hepatitis B virus infecting human choriocarcinoma JEG3 cells and its mechanism. Intervirology 2011; 54:276-81. [PMID: 21454957 DOI: 10.1159/000324528] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2010] [Accepted: 01/02/2011] [Indexed: 01/28/2023] Open
Abstract
AIM To build a hepatitis B virus (HBV)-infected human trophoblast cell model in vitro and determine the mechanism of intrauterine HBV infection. METHODS Serum from hepatitis B-infected patients containing HBV DNA >10(9) was drawn, subsequently inoculated into human trophoblast cells in vitro (JEG3) and passage-cultured. The supernatants and intracellular HBV viral load of inoculated cells were tested by real-time PCR, and HBV DNA was determined by Southern blot. RESULTS From inoculation of the 1st passage JEG3 cells, the supernatant viral load of the 1st passage was seen increasing over time, which peaked at 120 h, whereas the HBV viral load was seen decreasing gradually in subsequent passages, and tested negative after the 6th passage. In addition, infected cells of HBV DNA were tested by Southern blot, and showed continual expression in the subsequent cell passages 1-5 while passage 6 was negative. HBsAg was tested as positive from different passages 1-5, and its concentration was also seen decreasing with each subsequent passage cultured until the 6th passage when it tested negative. CONCLUSION HBV could infect human trophoblast cells (JEG3) in vitro, and it showed continual expression in subsequent cell passages 1-5.
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Affiliation(s)
- Yang Ding
- Infectious Disease Laboratory, China Medical University, Shengjing
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35
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Gao YT, Han T, Li Y, Yang B, Wang YJ, Wang FM, Jing X, Du Z. Enhanced specificity of real-time PCR for measurement of hepatitis B virus cccDNA using restriction endonuclease and plasmid-safe ATP-dependent DNase and selective primers. J Virol Methods 2010; 169:181-187. [PMID: 20691734 DOI: 10.1016/j.jviromet.2010.07.031] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2009] [Revised: 07/27/2010] [Accepted: 07/29/2010] [Indexed: 11/24/2022]
Abstract
The persistence of covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) in hepatocytes plays a key role in viral persistence and resistance to therapy. Therefore, quantitative cccDNA measurement is of clinical importance for evaluating the efficacy of antiviral drugs, selecting an appropriate treatment strategy, and predicting the prognosis. Current established methods for measurement of cccDNA need further improvement. A modified method was developed using digestion with restriction endonucleases that do not recognize sites in the HBV DNA and plasmid-safe ATP-dependent DNase (PSAD), and using a cccDNA-specific primer set in a real-time PCR reaction. The cccDNA-specific primer has a similar amplification efficiency as a commercial kit. Treatment of samples with restriction endonuclease followed by PSAD digestion increased significantly the specificity of a cccDNA-selective primer set compared with other treatments (P<0.05). Analysis of 35 serum and liver DNA samples from patients with hepatocellular carcinoma demonstrated that the amount of serum cccDNA is beyond the minimum detection limit and that the liver cccDNA quantity is about 0-49.2 copies/cell, consistent with previous reports. Taken together, this method has the potential for evaluating the efficacy of antiviral drugs.
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Affiliation(s)
- Ying-Tang Gao
- Key Laboratory of Artificial Cell, Institute for Hepatobiliary Disease, Tianjin Third Central Hospital, Tianjin, China
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36
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Detection of hepatitis B virus covalently closed circular DNA in paraffin-embedded and cryo-preserved liver biopsies of chronic hepatitis B patients. Eur J Gastroenterol Hepatol 2010; 22:952-60. [PMID: 20150816 DOI: 10.1097/meg.0b013e3283376a63] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may become an important predictor for treatment outcome or long-term follow-up. AIM To detect cccDNA in formalin-fixed, paraffin embedded (FFPE) and to compare with cryo-preserved liver tissue. METHODS Biopsies of 56 chronic hepatitis B patients were collected. Cryo-preserved and FFPE liver biopsies were available from 37 out of 56 patients. Paraffin was extracted with 1 ml xylene, followed by 100% alcohol and acetone. For the detection of cccDNA, selective primers were used. For quantification of hepatocytes a commercial Taqman beta-actin control kit was used. RESULTS The cccDNA was detected in 80% of FFPE and in 100% of cryo-preserved liver specimens. Recovery of hepatocytes and cccDNA was approximately a 100-fold lower in FFPE liver tissue, but intrahepatic cccDNA levels were comparable. In FFPE and cryo-preserved liver tissue, intrahepatic cccDNA levels correlated strongly with HBV DNA, hepatitis B e antigen (HbeAg), and plasma cccDNA levels. HbeAg positive chronic hepatitis B patients had significantly higher intrahepatic cccDNA levels compared with HBeAg negative patients (P<0.05). In HBeAg positive patients, no difference in intrahepatic cccDNA levels were seen between patients with active (histological activity index score>3; HBV DNA>20 000 IU/ml) and inactive hepatitis (histological activity index score</=3). In HBeAg negative chronic hepatitis B patients, intrahepatic cccDNA levels were significantly higher in patients with active hepatitis (P=0.004 and 0.001). CONCLUSION Recovery of hepatocytes and cccDNA in FFPE tissue was lower, but intrahepatic cccDNA in FFPE biopsies were comparable with cryo-preserved liver tissue. Therefore, FFPE liver tissue is an attractive alternative for cccDNA analysis when cryo-preserved tissue is not available.
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37
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Luan J, Yuan J, Li X, Jin S, Yu L, Liao M, Zhang H, Xu C, He Q, Wen B, Zhong X, Chen X, Chan HLY, Sung JJY, Zhou B, Ding C. Multiplex detection of 60 hepatitis B virus variants by maldi-tof mass spectrometry. Clin Chem 2009; 55:1503-9. [PMID: 19541863 DOI: 10.1373/clinchem.2009.124859] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
BACKGROUND Variations in the hepatitis B virus (HBV) genome may develop spontaneously or under selective pressure from antiviral therapy. Such variations may confer drug resistance or affect virus replication capacity, resulting in failure of antiviral therapy. METHODS A duplex PCR was used to amplify the region of the reverse transcriptase gene, the precore promoter, and the basal core promoter of the HBV genome. Four multiplex primer-extension reactions were used to interrogate 60 frequently observed HBV variants during antiviral therapy. Automated MALDI-TOF mass spectrometry (MS) was used for mutation detection. Capillary sequencing was used to confirm the MS results. RESULTS The limit of quantification was 1000 HBV copies/mL for multiplex detection of HBV variants. Fifty-three variants (88.3%) were analyzed successfully in at least 90% of the sera from 88 treatment-naive patients and 80 patients with virologic breakthrough. MS was able to detect twice as many minor variants as direct sequencing while achieving close to full automation. MS and direct sequencing showed only 0.1% discordance in variant calls. CONCLUSIONS This platform based on multiplex primer extension and MALDI-TOF MS was able to detect 60 HBV variants in 4 multiplex reactions with accuracy and low detection limits.
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Affiliation(s)
- Ju Luan
- Stanley Ho Centre for Emerging Infectious Diseases and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR
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38
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Takkenberg RB, Zaaijer HL, Molenkamp R, Menting S, Terpstra V, Weegink CJ, Dijkgraaf MGW, Jansen PLM, Reesink HW, Beld MGHM. Validation of a sensitive and specific real-time PCR for detection and quantitation of hepatitis B virus covalently closed circular DNA in plasma of chronic hepatitis B patients. J Med Virol 2009; 81:988-95. [PMID: 19382261 DOI: 10.1002/jmv.21477] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral replication and plays a role in persistence of HBV infection. The origin and significance of cccDNA in plasma however, is not well understood. A sensitive, specific, and reproducible real-time PCR for detection and quantitation of cccDNA in plasma of chronic hepatitis B patients was developed and validated. Four HBV DNA reference panels, and 96 plasma samples of chronic hepatitis B patients were analyzed. Results were compared with total HBV DNA levels, individual ALT levels and the Histology Activity Index (HAI). This cccDNA assay had a lower limit of detection at 15 copies/PCR, a lower limit of quantitation at 91 copies/PCR and a correlation coefficient (R) of 0.98 (P < 0.0001). cccDNA was detected in two of four international panels. Significant correlation was found between cccDNA and total HBV DNA levels in both panels (R = 0.96, and R = 0.43) and in samples of the chronic hepatitis B patients (R = 0.88, P < 0.0001). In 57% of these samples cccDNA was detectable. Mean level of cccDNA was 0.16% of total HBV load. Plasma cccDNA levels were higher in HBeAg positive samples than in HBeAg negative samples (4.91 log copies/ml vs. 3.88 log copies/ml, P < 0.0001). Levels of total HBV DNA and HBV genotype did not influence cccDNA detection. ALT levels and HAI-score were not correlated with plasma cccDNA levels. These findings suggest that cccDNA levels in plasma are not the result of increased hepatocyte degeneration, but indicate that other mechanisms might be responsible.
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Affiliation(s)
- R B Takkenberg
- AMC Liver Center, Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
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Mazet-Wagner AA, Baclet MC, Loustaud-Ratti V, Denis F, Alain S. Real-time PCR quantitation of hepatitis B virus total DNA and covalently closed circular DNA in peripheral blood mononuclear cells from hepatitis B virus-infected patients. J Virol Methods 2006; 138:70-79. [PMID: 16962180 DOI: 10.1016/j.jviromet.2006.07.019] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2006] [Revised: 07/12/2006] [Accepted: 07/19/2006] [Indexed: 12/29/2022]
Abstract
It remains unclear whether hepatitis B virus (HBV) replicates in extrahepatic tissues, and particularly in peripheral blood mononuclear cells (PBMCs), which may serve as a reservoir for the maintenance of infection. A real-time PCR assay for the quantitation of total and covalently closed circular (ccc) HBV DNA in serum and in PBMCs was developed. This assay was highly sensitive (detection limit: 27 IU/mL), linear over a wide range (9 log10), and was displayed high inter- and intra-assay reproducibility for the quantitation of total DNA. Genotypes A to E were detected and the results were consistent with those obtained with the COBAS Amplicor HBV Monitor Test. The specificity of the methodology was increased by prior treatment with an enzyme that digests relaxed circular DNA, and the elimination of background signals from virus adsorbed to the surface of PBMCs. HBV DNA was detected in the serum and PBMCs of 12 HBsAg-positive patients, with less than 1% in the cccDNA form. In conclusion, the quantitation of total and ccc HBV DNA in PBMCs is potentially useful as a non-invasive marker, and may help to increase our knowledge of the natural history of hepatitis B.
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Affiliation(s)
- A A Mazet-Wagner
- Laboratoire de Bactériologie-Virologie EA3175, Faculté de Médecine, Université de Limoges, 2 rue du Dr Marcland, 87000 Limoges, France
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40
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Yuen MF, Wong DKH, Sum SSM, Yuan HJ, Yuen JCH, Chan AOO, Wong BCY, Lai CL. Effect of lamivudine therapy on the serum covalently closed-circular (ccc) DNA of chronic hepatitis B infection. Am J Gastroenterol 2005; 100:1099-103. [PMID: 15842584 DOI: 10.1111/j.1572-0241.2005.41530.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
OBJECTIVE To determine the effect of 1-yr lamivudine treatment on serum covalently closed-circular DNA (cccDNA) level. PATIENTS AND METHOD Serum total HBV DNA and cccDNA levels at baseline, week 24, and week 52 were measured in 82 lamivudine-treated patients, 17 of whom received 1-yr placebo and acted as controls. RESULTS There was a significant reduction in the cccDNA levels from baseline (median 3.0 x 10(6) copies/ml) to week 24 (33,476 copies/ml) and week 52 (48,694 copies/ml) (p < 0.001 for both). The median reduction in cccDNA level at week 24 and 52 were 2.21 and 2.12 logs, respectively, which were significantly greater than those of controls (0.31 log, p < 0.001; 0.2 log, p < 0.001, respectively). Fifteen patients (18.3%) developed YMDD mutations by week 52. Compared to patients without YMDD mutations, patients with YMDD mutations had significantly less median reduction of total HBV DNA level (4.44 vs 3.65 logs, respectively, p= 0.02) and cccDNA level (2.27 vs 1.65 logs, respectively, p= 0.016) at week 24 and significantly less median reduction of cccDNA at week 52 (2.35 vs 0.8 logs respectively, p < 0.001). CONCLUSIONS One-year lamivudine treatment decreased serum cccDNA level by 2 logs. The chance of YMDD mutations at week 52 was related to the magnitude of viral suppression at week 24.
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Affiliation(s)
- Man-Fung Yuen
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China
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