Intrahepatic Total HBV DNA Research Articles

Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients

It has been proposed that serum hepatitis B core-related antigen (HBcrAg) reflects intrahepatic covalently closed circular (ccc)DNA levels. However, the correlation of HBcrAg with serum and intrahepatic viral markers and liver histology has not been comprehensively investigated in a large sample. We aimed to determine if HBcrAg could be a useful therapeutic marker in patients with chronic hepatitis B. HBcrAg was measured by chemiluminescent enzyme immunoassay in 130 (36 hepatitis B e antigen [HBeAg]+ and 94 HBeAg-) biopsy proven, untreated, patients with chronic hepatitis B. HBcrAg levels were correlated with: a) serum hepatitis B virus (HBV)-DNA, quantitative hepatitis B surface antigen and alanine aminotransferase levels; b) intrahepatic total (t)HBV-DNA, cccDNA, pregenomic (pg)RNA and cccDNA transcriptional activity (defined as pgRNA/cccDNA ratio); c) fibrosis and necroinflammatory activity scores. HBcrAg levels were significantly higher in HBeAg+ vs. HBeAg- patients and correlated with serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA and cccDNA levels, and transcriptional activity. Patients who were negative for HBcrAg (<3 LogU/ml) had less liver cccDNA and lower cccDNA activity than the HBcrAg+ group. Principal component analysis coupled with unsupervised clustering identified that in a subgroup of HBeAg- patients, higher HBcrAg levels were associated with higher serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA, cccDNA transcriptional activity and with higher fibrosis and necroinflammatory activity scores. Our results indicate that HBcrAg is a surrogate marker of both intrahepatic cccDNA and its transcriptional activity. HBcrAg could be useful in the evaluation of new antiviral therapies aiming at a functional cure of HBV infection either by directly or indirectly targeting the intrahepatic cccDNA pool. Hepatitis B virus causes a chronic infection which develops into severe liver disease and liver cancer. The viral covalently closed circular DNA (cccDNA) is responsible for the persistence of the infection in hepatocytes. To better manage patient treatment and follow-up, and to develop new antiviral treatments directly targeting the intrahepatic pool of cccDNA, serum surrogate markers reflecting the viral activity in the liver are urgently needed. In this work, we demonstrate that quantification of hepatitis B core-related antigen in serum correlates with cccDNA amount and activity and could be used to monitor disease progression.

Exonuclease I and III improve the detection efficacy of hepatitis B virus covalently closed circular DNA

BackgroundHepatitis 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. MethodsExonuclease 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. ResultsA 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). ConclusionsThe 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.

Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy

This study aimed to investigate the relationship between intrahepatic cccDNA and serum HBsAg in chronic Hepatitis B (CHB) patients with undetectable serum HBV DNA during antiviral therapy. We investigated HBsAg serum levels and their relationship to intrahepatic total cccDNA and HBV DNA in CHB patients with undetectable serum HBV DNA during oral antiviral therapy. Intrahepatic cccDNA and HBV DNA quantitation were performed in the same needle biopsy material, while serum HBsAg, HBeAg and HBV DNA levels were measured in samples drawn on the day of the liver biopsy. A total of 90 patients who had a liver biopsy were enrolled, including 80 patients with CHB and 10 patients with liver cirrhosis (LC). All the CHB patients were divided into HBeAg-positive and HBeAg-negative group. By using real-time PCR detection, we found that intrahepatic cccDNA and HBV DNA levels were higher in CHB patients than those in LC patients (Intrahepatic cccDNA: 6.15±1.19 vs. 6.12±0.36, HBV DNA: 7.26±0.49 vs. 5.59±0.45, both P<0.05). Intrahepatic cccDNA level was positively correlated with serum HBsAg in HBeAg-negative (r=0.66, P=0.02) and lower serum HBeAg (≤50S/CO) CHB patients (r=0.47, P=0.03), but not in higher serum HBeAg (>50S/CO) CHB patients (both P>0.05). In HBeAg negative patients, serum HBsAg level was correlated with intrahepatic total HBV DNA level (r=0.52, P=0.006). However, no relationship between HBsAg level and intrahepatic total HBV DNA level was found in HBeAg positive patients (both P>0.05). Serum HBsAg can be used to predict intrahepatic cccDNA and HBV DNA level in CHB patients with low serum HBeAg statues, especially in HBeAg negative patients.

Quantification of intrahepatic total HBV DNA in liver biopsies of HBV-infected patients by a modified version of COBAS® Ampliprep/COBAS®TaqMan HBV test v2.0

Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBAS®Ampliprep/COBAS®TaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R 2=0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R 2=0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBAS®Ampliprep/COBAS®TaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.

O011 : A nationwide survey of hepatitis E virus infection in liver transplant recipients in Japan

Results: Infrequent intrahepatic total HBVDNA (2/22, 9.1%) and cccDNA (1/22, 4.5%) were detected in biopsies; HBsAg and HBcAg IHC staining was negative. HBV-specific T cell responses were similar between “anti-HBc alone” individuals and HBV resolvers (Figure 1). Circulating HBV-memory B cell responses were detected in all “anti-HBc alone” individuals, consistent with an HBsAgspecific memory pool. After one HBV vaccine dose, increased antiHBs antibody levels were observed, accompanied by an expansion of HBsAg-specific memory B cells (P =0.0226) (Figure 2). Conclusions: “Anti-HBc alone” individuals showed HBV-specific T cell and memory B cell responses typical of previous viral exposure and protective memory, suggesting a resolved infection.

Effects of Entecavir on Hepatitis B Virus Covalently Closed Circular DNA in Hepatitis B e Antigen-Positive Patients with Hepatitis B

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.

Serum hepatitis B surface antigen is correlated with intrahepatic total HBV DNA and cccDNA in treatment‐naïve patients with chronic hepatitis B but not in patients with HBV related hepatocellular carcinoma

The aim of the study was to investigate correlations between intrahepatic hepatitis B virus total DNA, covalently closed circular DNA (cccDNA), and serum HBsAg in treatment-naïve chronic hepatitis B and HBV related hepatocellular carcinoma (HCC). Liver tissues were taken from 42 HBV related HCC and 36 patients with chronic hepatitis B. A fraction of DNA extracted from liver tissue was digested with a plasmid-safe ATP-dependent DNase and used for HBV cccDNA detection. The remaining DNA was used for the detection of HBV total DNA and β-globin, the latter of which is a housekeeping gene and quantified for normalization by real-time PCR. Quantitation of serum HBsAg was performed by a chemiluminescence assay. Serum HBsAg had positive correlations with serum HBV DNA (r = 0.636, P < 0.001), intrahepatic HBV total DNA (r = 0.519, P = 0.001) and cccDNA (r = 0.733, P < 0.001) in 36 treatment-naïve chronic hepatitis B, while HBsAg correlated poorly with DNA (r = 0.224, P = 0.210), intrahepatic total DNA and cccDNA in the tumor (r = 0.351, P = 0.031; r = 0.164, P = 0.324, respectively) and non-tumor (r = 0.237, P = 0.152; r = 0.072, P = 0.667, respectively) liver tissues of 42 HCC. HBV cccDNA and total DNA were significantly higher in liver tissue from chronic hepatitis B than in tumor and non-tumor of HCC (P < 0.001). Serum HBsAg and HBV DNA were also higher in chronic hepatitis B than in HCC (P < 0.001). It was concluded that levels of serum HBsAg and intrahepatic cccDNA and total DNA were significantly higher in chronic hepatitis B than in HCC, and significant correlations among them were observed in treatment-naïve chronic hepatitis B but not in HCC.

High HBV-DNA Titer in Surrounding Liver Rather Than in Hepatocellular Carcinoma Tissue Predisposes to Recurrence After Curative Surgical Resection

In this study the authors intended to investigate the relationship between intrahepatic hepatitis B virus (HBV)-DNA concentrations and posthepatectomy recurrence of HBV-associated hepatocellular carcinoma (HCC). High HBV-DNA level is strongly associated with HCC development in chronic HBV infection and considered to be a risk factor of HCC recurrence. A total of 109 patients with HBV-associated HCC who underwent curative surgical resection were followed up every 3 to 6 months for a median of 82 months. Intrahepatic total HBV-DNA titer was measured in HCC and surrounding liver tissues using a TaqMan probe-based real-time polymerase chain reaction method. HBV-DNA titers in HCC and surrounding liver were compared in accordance with patients' clinical, radiologic, and histopathological characteristics. The relationships between HBV-DNA titers in HCC or surrounding liver tissues and cumulative HCC recurrence rates were determined. Of the 109 patients, 67 (62%) showed posthepatectomy recurrence of HCC. In all patients, total HBV-DNA titers were significantly higher in HCCs than in surrounding liver tissues (P=0.019). HCC recurred more frequently in patients with higher than those with lower HBV-DNA titers in surrounding liver tissues (P=0.009). In contrast, the HCC recurrence rates were similar in patients with higher and those with lower HBV-DNA titers in HCC specimens (P=0.301). Multivariate analysis showed that tumor size >5 cm (P=0.008), the presence of portal vein thrombus (P=0.001), and high HBV-DNA titer in surrounding liver tissues (P=0.002) were independent risk factors for posthepatectomy HCC recurrence in patients with HBV-associated HCC. In patients with HBV-associated HCC, high HBV-DNA titer in surrounding liver rather than in the HCC itself is associated with posthepatectomy HCC recurrence after curative surgical resection.

Occult hepatitis B infection and HBV replicative activity in patients with cryptogenic cause of hepatocellular carcinoma

We aimed to investigate the incidence of occult hepatitis B infection (OBI) in patients with "cryptogenic" hepatocellular carcinoma (HCC) and to study the HBV replicative activity in these patients. Tumorous and adjacent nontumorous liver tissues were obtained from 33 cryptogenic HCC patients and 28 HCC patients with identifiable causes (13 with chronic hepatitis B [CHB], six with chronic hepatitis C, and nine alcohol-related). OBI was identified by nested polymerase chain reaction (PCR). Intrahepatic HBV DNA, covalently closed circular DNA (cccDNA), and pregenomic RNA (pgRNA) were quantified by real-time PCR and reverse-transcription PCR (RT-PCR), respectively. OBI was identified in 24 (73%) cryptogenic HCC patients, one (17%) HCC patient with HCV, and five (56%) patients with alcohol-related HCC. In HCC patients with OBI, HBV DNA were detected in ≥2 HBV genomic regions more often in nontumorous tissues than in tumorous tissues (90% versus 57%, respectively; P = 0.007). Cryptogenic HCC patients with OBI had lower intrahepatic total HBV DNA levels than HCC patients with CHB (median: 0.010 versus 3.19 copies/cell, respectively; P < 0.0001). Only six (26%) cryptogenic HCC patients with OBI had detectable cccDNA (median: <0.0002 copies/cell), which was significantly lower than that of the CHB patients (median: 0.005 copies/cell; P < 0.0001). HBV pgRNA were detectable in 12 (52%) cryptogenic HCC patients with OBI (median: 0.0001 copies/cell), which was significantly lower than that of the CHB patients (median: 2.90 copies/cell; P < 0.001). 73% of patients with apparently unidentifiable causes for HCC were HBV-related. The detection rate was higher in nontumorous tissues than tumorous tissues. The low intrahepatic HBV DNA and pgRNA levels indicated that persistent viral replication and possibly HBV integration are the likely causes of HCC in OBI patients.

Total and covalently closed circular DNA detection in liver tissue of long-term survivors transplanted for HBV-related cirrhosis

Background Life-long prophylaxis against HBV recurrence is recommended in patients transplanted for HBV-related disease. The risk of HBV reactivation is due to persistence of covalently closed circular (ccc) DNA in hepatocytes. Whether cccDNA persists in livers of long-term transplant survivors who received conventional prophylaxis is unknown. Aim To investigate the presence of intrahepatic total and cccDNA in transplanted patients with no evidence of biochemical markers of HBV recurrence. Methods Intrahepatic total and cccDNA were assessed using sensitive nested and real-time PCR from 44 HBsAg-positive patients (75% male; mean age 55.2 ± 8.9 years) who had undetectable serum HBV-DNA at transplant. The mean follow-up after transplant was 88.3 months (range, 18–159). Results One patient underwent HBV recurrence after transplant and was the only who tested positive for both intrahepatic total HBV-DNA and cccDNA. Of the 43 patients negative for all serological markers of HBV infection, only 2 tested positive for intrahepatic total HBV-DNA, but none for cccDNA. Conclusions Most patients with undetectable HBV-DNA at transplant, who received conventional HBV prophylaxis, have no evidence of intrahepatic total HBV-DNA and cccDNA. cccDNA should be considered a new additional diagnostic tool, also to identify patients at low risk of HBV recurrence after liver transplantation.

HBsAg Seroclearance in Chronic Hepatitis B in Asian Patients: Replicative Level and Risk of Hepatocellular Carcinoma

Our aims were to study the virologic, histologic, and clinical outcome in chronic hepatitis B (CHB) patients with hepatitis B surface antigen (HBsAg) seroclearance. We determined the age of HBsAg seroclearance that is associated with a lower risk for hepatocellular carcinoma (HCC) in 298 CHB patients (median follow-up, 108 months). The following virologic and histologic features were also determined: liver stiffness (n = 229), liver histology, serum HBV DNA levels over time (n = 265), intrahepatic HBV DNA with covalently closed circular DNA (cccDNA) levels, and messenger RNA (mRNA) expression. The median age of HBsAg seroclearance was 49.6 years. Seven (2.4%) patients developed HCC. Cumulative risk for HCC was higher in patients with HBsAg seroclearance at ages >or=50 years compared with those with HBsAg seroclearance at ages <50 (P = .004) years. Of these 2 groups of patients, 29.5% and 7.9%, respectively, had significant fibrosis by liver stiffness measurement (P = .001), and 15.4% of patients had mild histologic fibrosis. Intrahepatic total HBV DNA and cccDNA were detected in 100% and 79.3% of patients, respectively. All patients had undetectable surface and precore/pregenomic RNA transcripts. One (9.1%) patient had X mRNA expression. Serum HBV DNA were detectable in 13.4%, 6.1%, and 3.7% of patients within 1 year and 5-10 and >10 years after HBsAg seroclearance, respectively, and 82.1% patients had persistently normal alanine aminotransferase levels. HBV persisted at low replicative and transcriptional levels after HBsAg seroclearance. HBsAg seroclearance at age <50 years was associated with a lower risk for the development of HCC.

Lower viral production in HBeAg-negative patients is due to both reduced pool size and activity of the HBV cccDNA

Background/Aim: Chronic infection with hepatitis B virus (HBV) is due to maintenance of covalently closed circular DNA (cccDNA) and knowledge of factors regulating cccDNA pool size and viral production may help understanding mechanisms of viral clearance. To evaluate activity of cccDNA in HBV chronically infected (CH-B) patients, we analyzed serological parameters (HBV-DNA titres and HBsAg), as well as intrahepatic total HBV-DNA and cccDNA levels in liver biopsies obtained from 120 patients in different phases of chronic HBV infection.

Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is responsible for persistent infection of hepatocytes. The aim of this study was to determine changes in intrahepatic cccDNA in patients with chronic hepatitis B (CH-B) during 48 weeks of antiviral therapy and its correlation to virological, biochemical, and histological parameters. Twenty-six HBsAg-positive CH-B patients received combination treatment with pegylated interferon alpha-2b (peg-IFN) and adefovir dipivoxil (ADV) for 48 weeks. Paired liver biopsies from before and at the end of treatment were analyzed for intrahepatic HBV-DNA. Median serum HBV-DNA had decreased by -4.9 log10 copies/mL at the end of treatment and was undetectable in 13 individuals (54%). Median intrahepatic total HBV-DNA and cccDNA had decreased by -2.2 and -2.4 log10, respectively. Changes in intracellular HBV-DNA positively correlated with HBsAg serum reduction and were accompanied by a high number of serological responders. Eight of 15 HBeAg-positive patients lost HBeAg, and five developed anti-HBe antibodies during treatment. These eight patients exhibited lower cccDNA levels before and at the end of therapy than did patients without HBeAg loss. Four patients developed anti-HBs antibodies. ALT normalized in 11 patients. The number of HBs-antigen- and HBc-antigen-positive hepatocytes was significantly lower after treatment, suggesting the involvement of cytolytic mechanisms. In conclusion, combination therapy with peg-IFN and ADV led to marked decreases in serum HBV-DNA and intrahepatic cccDNA, which was significantly correlated with reduced HBsAg.

Quantitative Analysis of Hepatic CCCDNA during Combination Therapy of Peginterferon ALFA 2B Plus Adefovir Dipivoxil in Patients with Chronic Hepatitis B: Interim Analysis of a single Center Pilot Study

Background: Hepatitis B Virus cccDNA is the replicative intermediate in the nuclei of infected hepatocytes that is responsible for viral persistence during chronic HBV infection and antiviral therapy. The aims of this study were 1) to determine changes in intrahepatic cccDNA levels in paired biopsy samples from patients during therapy with Peginterferon alfa 2b and adefovir dipivoxil (primary endpoint) using a quantitative PCR assay (Werle, Gastroenterology, in press) and 2) to study the safety and efficacy of the combination therapy for 48 weeks. Methods: 17 of 26 patients completed the 48 week course of combination therapy as of March 2004. Baseline characteristics: 9 patients were HBeAg positive, 8 were negative for HBeAg, median serum HBV DNA 2×106 copies/ml (PCR, LLQ 200 copies/ml), median ALT 3.1 xULN, median total intrahepatic HBV DNA 262 copies/hepatocyte and median intrahepatic cccDNA 2.8 molecules/hepatocyte. Results: 2 patients seroconverted to HBs antibodies, 2 additional patients showed HBe seroconversion. In 10/17 patients (58%) serum HBV DNA had become undetectable (<200 copies/mL). 7/17 patients were still PCR-positive with a median serum HBV DNA at week 48 of 1×102 copies/ml (–4.3log10 copies/mL reduction from baseline). ALT normalized in 9/17 (52%), improved in 5/17 (30%), and slightly increased in 3/17 patients (18%). One patient missed week 48 biopsy, accounting for 16 paired biopsies. Median intrahepatic total HBV DNA at week 48 was 2.1 copies/hepatocyte (–2.1log). Intrahepatic cccDNA was undetectable in 9/16 (56%), and still positive in 7/16 biopsies including 3 patients with unaffected cccDNA levels and 4 patients with increased intrahepatic cccDNA (max. x48). Except for one patient, persistence of cccDNA was accompanied by elevated ALT level (max. 1.7 xULN) and/or detectable serum HBV DNA. 10/16 (63%) patients showed an improvement in the METAVIR score, either in inflammation and/or fibrosis. No unexpected adverse events were reported. Conclusion: In this on-going small study cohort, administration of Peginterferon alfa 2b once weekly and adefovir dipivoxil 10mg daily was safe and well tolerated. Intrahepatic cccDNA copy numbers are reduced to undetectability (<3 copies/10.000 cells) in the majority of patients.