Quantitative Analysis of HBV cccDNA from Clinical Specimens
Attempts to investigate changes in various forms of intrahepatic hepatitis B virus (HBV) DNA during antiviral therapy have been hampered by limitations in technologies and scarcity of adequate tissue for analysis. We used a sensitive, specific assay to detect and quantitate covalently closed circular DNA (cccDNA) from total intrahepatic HBV DNA in clinical liver specimens. Total HBV DNA and cccDNA from 21 needle-biopsy specimens were quantified, with levels ranging from 0.1 to 9.8 copies/cell and 0.3 to 491.0 copies/cell, respectively. Then, we performed the same determinations on baseline and week-52 liver needle-biopsy specimens from eight patients enrolled in a clinical trial and evaluated the association between intrahepatic HBV DNA levels and serological and virological endpoints. In most patients, levels of intrahepatic HBV DNA, including cccDNA, decreased over the 52-week study, regardless of therapy or serological outcome. Higher ratios of cccDNA to total HBV DNA were detected at week 52 than at baseline indicating a shift in predominance of nonreplicating virus in posttreatment specimens. In patients who achieved treatment-related or spontaneous hepatitis B e antigen (HBeAg) responses, including those harbouring tyrosine-methionine-aspartate-aspartate-mutant HBV, levels of intrahepatic and serum HBV DNA suppression were greater than those in patients without HBeAg responses. In conclusion, this pilot study of intrahepatic HBV replicative forms in patients with chronic hepatitis B indicated that total intrahepatic and, specifically, cccDNA levels are not static but change as a reflection of serological and virological events.
Antiviral therapy of chronic hepatitis B can lead to suppression of serum HBV DNA, hepatitis B e antigen (HBeAg) seroconversion, normalization of serum aminotransferase activity and improvement in liver histology; however, traces of hepatitis B virus (HBV) DNA can be detected for decades after HBeAg and hepatitis B surface antigen (HBsAg) seroconversion, i.e. even after resolution of infection. Because HBV DNA exists in different forms, some of which can be maintained for protracted periods, perhaps indefinitely, in infected patients, an appreciation of the viral replication cycle is critical to understanding the impact of low-level viral replication on the natural history of chronic hepatitis B.
The HBV genome is a partially double-stranded DNA molecule. Upon infection, this genomic form is converted in the nucleus to a covalently closed circular DNA (cccDNA) molecule, which then serves as the template for transcription of viral RNA and DNA replication. New copies of the partially double-stranded DNA genome can be incorporated into virions as they are assembled in the cytoplasm and secreted or cycled directly back to the nucleus, thus replenishing cccDNA pools. By maintaining cccDNA pools in the nucleus, this intracellular amplification pathway ensures maintenance of viral infection.
Until recently, studies of the relation between cccDNA and disease status/clinical outcome have been hampered by technological limitations. Initial assays for cccDNA relied upon selective extraction of nonprotein-bound DNA followed by Southern blotting. This technique is well suited for analysing cccDNA in animal models, in which large volumes of infected tissue are available and levels of virus are very high, but of limited utility in clinical settings, in which only limited amounts of tissue (e.g. needle biopsies) are available. Polymerase chain reaction (PCR)-based methods that provide enhanced sensitivity reduce the amount of tissue needed and are more applicable to clinical settings. The main disadvantage of all PCR-based methodologies, however, is the inability to ensure selective amplification of the different forms of HBV DNA.
Köck and Schlicht developed a PCR-based technique to detect HBV DNA and distinguish HBV cccDNA from partially double-stranded, relaxed circular DNA (rcDNA) using primer pairs that amplify across the gapped regions in the rcDNA template, thereby selectively amplifying cccDNA. Stoll-Becker et al. modified the PCR-based method to include digestion with mung bean nuclease, which removes single-stranded DNA in the rcDNA form of HBV, reducing the possibility that rcDNA can serve as a template for cccDNA-specific primer pairs. Using this modified PCR-based method, these investigators detected cccDNA in peripheral blood mononuclear cells from highly viraemic, HBV-infected patients. Real-time detection platforms increase the sensitivity of PCR assays compared with gel-based PCR assays, and 'real-time PCR' is now considered the gold standard for selective quantitation of cccDNA from clinical biopsy specimens.
New techniques that have become available recently have sufficient sensitivity and selectivity to discriminate cccDNA from total intrahepatic HBV DNA in liver-biopsy specimens. Using one of these techniques, we conducted a pilot study to assess the levels of total HBV DNA and cccDNA within sequentially obtained, snap-frozen liver-biopsy specimens from patients receiving therapy with lamivudine, which is followed by lamivudine plus interferon-α or placebo. Results were correlated with serum HBV DNA levels, mutant or wild-type status of the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV reverse transcriptase and serological outcome after 52 weeks of therapy.
Summary and Introduction
Summary
Attempts to investigate changes in various forms of intrahepatic hepatitis B virus (HBV) DNA during antiviral therapy have been hampered by limitations in technologies and scarcity of adequate tissue for analysis. We used a sensitive, specific assay to detect and quantitate covalently closed circular DNA (cccDNA) from total intrahepatic HBV DNA in clinical liver specimens. Total HBV DNA and cccDNA from 21 needle-biopsy specimens were quantified, with levels ranging from 0.1 to 9.8 copies/cell and 0.3 to 491.0 copies/cell, respectively. Then, we performed the same determinations on baseline and week-52 liver needle-biopsy specimens from eight patients enrolled in a clinical trial and evaluated the association between intrahepatic HBV DNA levels and serological and virological endpoints. In most patients, levels of intrahepatic HBV DNA, including cccDNA, decreased over the 52-week study, regardless of therapy or serological outcome. Higher ratios of cccDNA to total HBV DNA were detected at week 52 than at baseline indicating a shift in predominance of nonreplicating virus in posttreatment specimens. In patients who achieved treatment-related or spontaneous hepatitis B e antigen (HBeAg) responses, including those harbouring tyrosine-methionine-aspartate-aspartate-mutant HBV, levels of intrahepatic and serum HBV DNA suppression were greater than those in patients without HBeAg responses. In conclusion, this pilot study of intrahepatic HBV replicative forms in patients with chronic hepatitis B indicated that total intrahepatic and, specifically, cccDNA levels are not static but change as a reflection of serological and virological events.
Introduction
Antiviral therapy of chronic hepatitis B can lead to suppression of serum HBV DNA, hepatitis B e antigen (HBeAg) seroconversion, normalization of serum aminotransferase activity and improvement in liver histology; however, traces of hepatitis B virus (HBV) DNA can be detected for decades after HBeAg and hepatitis B surface antigen (HBsAg) seroconversion, i.e. even after resolution of infection. Because HBV DNA exists in different forms, some of which can be maintained for protracted periods, perhaps indefinitely, in infected patients, an appreciation of the viral replication cycle is critical to understanding the impact of low-level viral replication on the natural history of chronic hepatitis B.
The HBV genome is a partially double-stranded DNA molecule. Upon infection, this genomic form is converted in the nucleus to a covalently closed circular DNA (cccDNA) molecule, which then serves as the template for transcription of viral RNA and DNA replication. New copies of the partially double-stranded DNA genome can be incorporated into virions as they are assembled in the cytoplasm and secreted or cycled directly back to the nucleus, thus replenishing cccDNA pools. By maintaining cccDNA pools in the nucleus, this intracellular amplification pathway ensures maintenance of viral infection.
Until recently, studies of the relation between cccDNA and disease status/clinical outcome have been hampered by technological limitations. Initial assays for cccDNA relied upon selective extraction of nonprotein-bound DNA followed by Southern blotting. This technique is well suited for analysing cccDNA in animal models, in which large volumes of infected tissue are available and levels of virus are very high, but of limited utility in clinical settings, in which only limited amounts of tissue (e.g. needle biopsies) are available. Polymerase chain reaction (PCR)-based methods that provide enhanced sensitivity reduce the amount of tissue needed and are more applicable to clinical settings. The main disadvantage of all PCR-based methodologies, however, is the inability to ensure selective amplification of the different forms of HBV DNA.
Köck and Schlicht developed a PCR-based technique to detect HBV DNA and distinguish HBV cccDNA from partially double-stranded, relaxed circular DNA (rcDNA) using primer pairs that amplify across the gapped regions in the rcDNA template, thereby selectively amplifying cccDNA. Stoll-Becker et al. modified the PCR-based method to include digestion with mung bean nuclease, which removes single-stranded DNA in the rcDNA form of HBV, reducing the possibility that rcDNA can serve as a template for cccDNA-specific primer pairs. Using this modified PCR-based method, these investigators detected cccDNA in peripheral blood mononuclear cells from highly viraemic, HBV-infected patients. Real-time detection platforms increase the sensitivity of PCR assays compared with gel-based PCR assays, and 'real-time PCR' is now considered the gold standard for selective quantitation of cccDNA from clinical biopsy specimens.
New techniques that have become available recently have sufficient sensitivity and selectivity to discriminate cccDNA from total intrahepatic HBV DNA in liver-biopsy specimens. Using one of these techniques, we conducted a pilot study to assess the levels of total HBV DNA and cccDNA within sequentially obtained, snap-frozen liver-biopsy specimens from patients receiving therapy with lamivudine, which is followed by lamivudine plus interferon-α or placebo. Results were correlated with serum HBV DNA levels, mutant or wild-type status of the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV reverse transcriptase and serological outcome after 52 weeks of therapy.
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