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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study examined a signal amplification assay, the Invader assay, for the quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in liver biopsies and sera. DNA was extracted from liver biopsy and serum samples were collected from 16 hepatitis B e antigen (HBeAg)-positive and 36 antibody-to-HBeAg-positive (anti–HBe-positive) chronic hepatitis B patients. The amount of total HBV DNA and cccDNA was measured using the Invader assay. Anti–HBe-positive patients had lower median total intrahepatic HBV DNA (P < .001) and intrahepatic cccDNA levels (P = .001) than HBeAg-positive patients. Intrahepatic cccDNA correlated positively with the total intrahepatic HBV DNA (r = 0.950, P < .001). However, the proportion of intrahepatic HBV DNA in the form of cccDNA was inversely related to the amount of total intrahepatic HBV DNA (r = −0.822, P < .001). A small amount of cccDNA was detected in 39 of 52 (75%) serum samples. Anti-HBe-positive patients had lower median serum cccDNA levels than HBeAg-positive patients (P = .002). Serum HBV DNA correlated positively with intrahepatic total HBV DNA (r = 0.778, P < .001) and intrahepatic cccDNA (r = 0.481, P = .002). In conclusion, the Invader assay is a reliable assay for the quantitation of cccDNA. Serum and intrahepatic total HBV DNA and cccDNA levels become lower as the disease progresses from HBeAg-positive to anti–HBe-positive phase, with cccDNA becoming the predominant form of intrahepatic HBV DNA. (HEPATOLOGY 2004;40:727–737.)

Chronic hepatitis B virus (HBV) infection affects approximately 400 million people in the world.1 One crucial step in the HBV life cycle is the formation of a covalently closed circular form of the viral genome through DNA repair of the relaxed circular replicative HBV DNA inside the nuclei of hepatocytes. Covalently closed circular DNA (cccDNA) provides the template for viral and pregenomic messenger RNA. In vitro studies have shown that lamivudine has a profound effect on relaxed circular DNA (rcDNA) while having little or no effect on cccDNA.2, 3 This is one possible reason for the rebound of HBV DNA to pretreatment levels often seen after lamivudine withdrawal.4

Another nonreplicative form of HBV DNA is the double-stranded linear (DL) form produced by in situ priming during HBV replication.5 DL DNA is a possible precursor to HBV DNA integration.6–8 It can also form cccDNA through nonhomologous recombination at its ends via a process called illegitimate replication.9, 10

cccDNA monitoring and the development of an accurate quantitative assay for cccDNA are becoming important in the understanding of the natural history and management of chronic hepatitis B (CHB). Most attempts for the quantitation of cccDNA have been made with liver biopsies from ducks or woodchucks.11–16 Quantitation of cccDNA in human peripheral blood mononuclear cells and liver biopsies has been performed.17–20 In these studies, primers spanning across the gap in the minus strand and corresponding to the variable region on the plus strand were used to amplify across noninterrupted cccDNA. It should be noted that, even with selective polymerase chain reaction (PCR) methods, amplification of residual rcDNA can still occur after self-annealing of the partial elongation products. The problem of nonspecific amplification of rcDNA is a major obstacle in the development of an accurate cccDNA quantitation assay. A non-PCR assay, the Invader assay, has been developed for the quantitative detection of HBV cccDNA.21 This study aimed to verify the validity of the Invader assay and to employ this assay to quantify cccDNA in liver biopsies and sera from CHB patients in different stages of disease.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients.

This study was approved by the Ethics Committee at the University of Hong Kong. Liver biopsies were collected from 16 hepatitis B e antigen (HBeAg)-positive patients, 36 antibody-to-HBeAg-positive (anti–HBe-positive) untreated CHB patients, and 5 non–HBV patients (3 with chronic hepatitis C, 1 with unexplained liver function derangement, and 1 with suspected porphyria) acting as controls. Serum samples were collected within 1 week of liver biopsies.

DNA Extraction and Serum HBV DNA Quantitation.

Total DNA was extracted from needle liver biopsies (0.5–1.5 cm) and 200 μL of sera using the QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany). Serum total HBV DNA was measured by the COBAS AMPLICOR HBV MONITOR Test (COBAS-AM assay) (Roche Diagnostics, Branchburg, NJ). Liver-free HBV DNA purification, to eliminate human genomic DNA (hgDNA), was performed by a modified Hirt procedure22 as described by Lee et al.23

DNA Quantitation by the Invader Assay.

Total HBV DNA, cccDNA, and hgDNA were quantified by the Invader HBV DNA assay (Third Wave Technologies, Madison, WI). The principle of the Invader assay has been described21 and is illustrated in Fig. 1. Briefly, two oligonucleotides, the primary probe and Invader oligonucleotide, hybridized to the target DNA to form a partially overlapping structure, which was cleaved by a Cleavase enzyme to generate an oligonucleotide called a 5′-flap. At a specific reaction temperature, the primary probe cycles on the target DNA, generating released 5′-flaps, which were amplified proportionally to the concentration of the target DNA. Fluorescence resonance energy transfer cassettes were used to react with the released flaps and generate a fluorescence signal measurable with real-time PCR machines.

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Figure 1. Schematic diagram of the Invader assay, using Invader probe set MS1 as an example [diagram modified from Kwiatkowski et al.21]. In the fluorescence resonance energy transfer (FRET) cassette, F represents the fluorophore reporter dye and Z represents the quencher.

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Three sets of oligonucleotides were used: minus strand (MS) 1 for HBV DNA MS (hence total HBV DNA) detection, plus strand (PS) 2 for cccDNA detection, and hgDNA for human insulin-like growth factor 1 gene detection (Fig. 2). Both the MS1 and PS2 sets hybridize to the HBV DNA sequences near the direct repeat 2 (DR2) element. Because the Cleavase site for set PS2 is at the 5′ base of DR2, cleavage is possible only if a covalent linkage of the 3′ end of the plus strand to the 5′ base of DR2 is present, as in cccDNA (Fig. 3).

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Figure 2. Invader probe sets and synthetic oligonucleotides used in this study. (A) DNA sequence and alignment of the 4 synthetic oligonucleotides and the 2 Invader probe sets used in the synthetic oligonucleotide target specificity experiments. The Invader probe sets PS2 and MS1 are aligned above and below the synthetic oligonucleotides, respectively. The PS template (oligo #3) and MS template (oligo #4) oligonucleotides represent the intact plus and minus strands of the HBV genome around the DR2 region, respectively. The PS probe oligo complement (oligo #1) contains a sequence identical to the HBV PS sequence just upstream of the DR2 element, whereas the PS Invader oligo complement (oligo #2) contains a sequence identical to the HBV PS sequence immediately downstream of the DR2 element. The nucleotides in the synthetic oligonucleotides corresponding to the DR2 element are capitalized. (B) Invader probe set used for human genomic DNA determination. The numbers in the template refer to those in the human chromosome 12q23 human insulin-like growth factor 1 complementary DNA sequence in GenBank (accession number M37484). PS2, plus strand 2; MS, minus strand 1; hgDNA, human genomic DNA.

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Figure 3. Detection of HBV DNA plus and minus strands with the probe sets PS2 and MS1, respectively, using the Invader assay. The relative hybridization positions of the probe sets are shown in the native (left) and denatured (right) forms of the HBV genome. Only the region around the DR2 element is shown in the denatured form. CCC, covalently closed circular; DR1, direct repeat 1; DR2, direct repeat 2; PS2, plus strand 2; MS, minus strand 1; RC, relaxed circular.

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Five microliters of DNA extracted from the sera and liver biopsies, diluted to 15 μL, were denatured at 95°C for 5 minutes and rapidly quenched. Five microliters of a reaction master mix, containing 40 mmol/L Mops (pH 7.5), 50 mmol/L MgCl2, 2 μmol/L primary probe, 2 μmol/L Invader oligonucleotide, 1 μmol/L fluorescence resonance energy transfer cassette, and 50 ng Cleavase, was added to each denatured sample. The reaction mixtures were then incubated in the Rotor-Gene 3000 Real-time Cycler (Corbett Research, Mortlake, Australia) with the following temperature setting: 80°C for 2 minutes, followed by a single temperature incubation at 64°C for 240 minutes, with fluorescence signal collection at 1-minute intervals.

Signal generation of the Invader assay follows a quadratic kinetics, and there is a linear relationship between the target copy level and the quadratic coefficient.24 For each reaction, the quadratic coefficient of the generation of fluorescent signal was determined. The amounts of total HBV DNA and cccDNA in a sample were determined via extrapolation from standard curves of the quadratic coefficients generated by reactions on external plasmid standards of known concentrations, using the probe sets MS1 and PS2, respectively. Intrahepatic total HBV DNA and cccDNA levels were normalized by the amount (in nanograms) of hgDNA in the samples, as determined using human insulin-like growth factor 1 detection. Cell numbers were calculated based on an estimated 6.667 pg hgDNA per cell.

Synthetic Oligonucleotide Target Specificity Assay.

Synthetic oligonucleotides were used to assess the cccDNA specificity of the PS2 probe set. The sequences and the alignments of the oligonucleotides and the Invader probe sets MS1 and PS2 are shown in Fig. 2. The oligonucleotides are designed to mimic the DNA sequence arrangement at DR2. The PS (oligo #3) and MS (oligo #4) templates resemble the complete plus and minus strands at DR2, respectively. The PS Invader oligo complement (oligo #2) represents the 5′ end of the HBV plus strand, and the PS primary probe oligo complement (oligo #1) represents the plus strand sequence upstream of DR2. The experimental design is shown in Fig. 4. In the initial enzyme digestion reaction, equal molar (100 nmol/L) of oligos #1, #2, and #4 were incubated with 100 ng of Cleavase and 2U T4 DNA ligase at 37°C. An intact DNA duplex is generated only if both Cleavase and ligase are present; absence of either one would render no linkage of oligos #1 and #2.

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Figure 4. Schematic diagram for the principle of the synthetic oligonucleotide target specificity assay. The sequences of the DR2 element are capitalized. Oligo #2P represents the 5′ phosphorylated oligo #2. MS, minus strand 1; PS2, plus strand 2.

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The reaction mixture was then diluted and subjected to HBV PS and MS detection using the Invader assay. The result was expressed as the ratio of the sample quadratic coefficient to the quadratic coefficient obtained from the no template control reaction, designated as fold-over-zero (FOZ). Figure 5A illustrated the expected result when different components were used. Conditions I and II were the positive controls, in which both the PS and MS templates were incubated with or without enzymes. Only the reactions with intact PS templates (conditions I and II) or with both Cleavase and ligase (condition VI) could produce a positive PS signal. Without one or both of the enzymes, no signal would be generated by the Invader probe set PS2.

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Figure 5. Results of the synthetic oligonucleotide assay. (A) Schematic representation of the expected results for the different reaction conditions (I–VI), as shown by the positive (+) and negative (−) signs when either Invader probe set MS1 or PS2 was used. The substrates used in the reactions, as shown by the thick colored bars, were incubated in the appropriate reaction buffer with or without enzymes. The enzymes used in the reaction conditions were as follows: I, no enzyme was added; II, both ligase and Cleavase were added; III, no enzyme was added; IV, only ligase was added; V, only Cleavase was added; VI, both ligase and Cleavase were added. The Invader probe sets MS1 and PS2 are indicated by the thin colored arrows. (B) Actual fluorescence signal result. The four control oligonucleotides (#1–#4) are the same as those in Fig. 2. In the 4 control reactions, no signal (FOZ<1) was generated by either probe set MS1 or PS2 when either oligo #1 or oligo #2 alone was used as a template. Invader reaction with probe set PS2 and a PS template (oligo #3) generated an average signal FOZ of 12.65, while the average signal FOZ was 0.91 with probe set MS1. In contrast, the MS template (oligo #4) generated an average signal FOZ of 21.87 with probe set MS1, whereas no signal was produced with probe set PS2 (FOZ = 0.83). MS, minus strand 1; PS2, plus strand 2; DR2, direct repeat 2; L, ligase; C, cleavase.

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S1 Nuclease Treatment.

S1 nuclease was used to degrade single-stranded DNA in the liver biopsies and sera, leaving only intact double-stranded DNA. DNA extracted from 2 liver biopsies and 4 serum samples were treated with 1U S1 nuclease at 37°C for 5 minutes, 30 minutes, and 1 hour and 10 U and 50 U S1 nuclease for 1 hour. The reaction mixtures were purified using the QIAquick PCR purification kit (QIAGEN GmbH). As negative controls, samples were incubated at the same conditions without enzyme.

Purification of HBV Virions and PCR Detection of HBV DNA.

HBV virions were purified from sera using polyethylene glycol (PEG8000), followed by digestion with deoxyribonuclease and ribonuclease as suggested.25, 26 PCR was used to detect both total HBV DNA and cccDNA in virions. For total HBV DNA detection, 20 pmol of primers LQZ105 (5′-TCGCTGGATGTGTCTGCGGCGTTTTAT-3′) and LQZ106 (5′-TAGAGGACAAACGGGCAACATACC-3′) were used to amplify a 112-bp S-gene fragment with the following conditions: 95°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Two previously published nested PCR methods were used to detect cccDNA.20, 27 All PCR products were analyzed in agarose gels.

Invader Assay Intra-assay and Interassay Variation Experiments.

All samples were analyzed in duplicate. To assess interassay variation, DNA extracted from 3 HBeAg-positive and 3 anti–HBe-positive liver biopsies with both low and high intrahepatic HBV DNA levels were repeated in 4 independent Invader reactions on 4 consecutive days.

Histology Assessment.

Histological sections of liver biopsy specimens were stained with hematoxylin-eosin. Based on a modified Knodell scoring system,28 the degree of necroinflammation was classified into the following categories: minimal, mild, moderate, and marked. The degree of fibrosis was classified as none, mild, moderate, or cirrhosis.

Statistical Analysis.

Statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 11.0 for Windows, SPSS, Chicago, IL). Continuous variables were tested for normality using the Kolmogorov-Smirnov test. Normally distributed variables were tested using the Student's t test, whereas continuous variables with skewed distribution were tested using the Mann-Whitney U test. Correlation between two variables was tested using Pearson's correlation analysis after logarithmic transformation of data with skewed distributions. Categorical variables were tested using the chi-squared test or Fisher's exact test. Statistical significance was denoted as a P value less than .05.

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Demographic, Serological, and Histological Data.

The demographic, serological, and histological data for the 52 CHB patients are listed in Table 1. Anti–HBe-positive patients had a significantly higher mean age than HBeAg-positive patients (P = .009) at the time of liver biopsy. There was no significant difference between the anti–HBe-positive and HBeAg-positive patients in the median alanine aminotransferase levels (P = .606) and median bilirubin levels (P = .2), nor in the degree of necroinflammation and fibrosis (necroinflammation: minimal/mild vs. moderate/marked, P = .085; fibrosis: none/mild vs. moderate/cirrhosis, P = .56).

Table 1. Demographic, Serological, and Histological Data
 HBeAg-positiveAnti–HBe-positive
Number of patients1636
Sex ratio (male/female)11:531:5
Mean age at biopsy ± SD (y)35.8 ± 10.743.9 ± 9.5
Median alanine aminotransferase level (range), U/L73 (38–357)97 (21–576)
Median bilirubin alanine aminotransferase level (range), μmol/L16 (4–77)12 (5–28)
Histological data  
 Necroinflammation  
  Minimal314
  Mild1315
  Moderate07
  Marked00
 Fibrosis  
  None08
  Mild69
  Moderate917
  Cirrhosis12

Specificity of the Invader Assay.

The result of the synthetic oligonucleotide test is shown in Fig. 5B. The PS2 probe oligo complement, PS2 Invader oligo complement, PS template, and MS template (oligos #1–#4, respectively) were included as controls. All 6 test reaction conditions (I–VI) contained an intact MS template, and all produced significant signal FOZs with the Invader probe set MS1. Conditions I and II, as well as the PS template control, contained intact PS templates, and the average signal FOZs of the Invader reactions with the PS2 probe set were 12.65, 13.14, and 12.65, respectively. In conditions III, IV, and V, either one or both of the Cleavase and ligase was missing, and significant signal could only be produced with probe set MS1, but not with probe set PS2. Condition VI produced an average signal FOZ of 12.61, indicating the formation of intact PS templates. This result demonstrated that the Invader probe set PS2 is highly specific for an intact, uninterrupted plus-stranded HBV DNA sequence and hence is specific for cccDNA.

As shown in Table 2, nuclease treatment did not reduce the total HBV DNA amount in the plasmid standard (P1), while the ratio of cccDNA to total HBV DNA remained at 100%, indicating the supercoiled plasmid standard can withstand the digestion conditions used. As the incubation time and amount of S1 nuclease increased, the 2 biopsies and 4 serum samples had a gradual reduction of total HBV DNA and a gradual increase in the cccDNA/total HBV DNA ratio. When 50 U S1 nuclease was used, the amount of rcDNA in the samples was further reduced, and homogenous samples of 100% cccDNA were generated.

Table 2. Total HBV DNA and Percentage of cccDNA Over Total HBV DNA in Biopsy and Serum Samples at Different Duration of S1 Nuclease Treatment
Sample No.S1 Nuclease Unit UsedTreatment Time (min)Total HBV DNA/mL DNA Extract, log10 (copies/mL)cccDNA Over Total HBV DNA, %
P1 (plasmid standard)1U07.25100
 1U57.27100
 1U307.65100
 1U607.61100
 10U607.50100
 50U607.37100
B1 (liver biopsy)1U07.644.19
 1U57.4512.3
 1U306.6162.6
 1U606.7059.3
 10U606.1860.8
 50U605.85100
B2 (liver biopsy)1U08.2310.4
 1U57.8829.7
 1U307.3285.6
 1U607.2894.1
 10U606.8597.8
 50U606.91100
S1 (serum)1U08.613.61
 1U58.589.09
 1U308.1127.9
 1U607.7647.3
 10U606.8549.3
 50U607.01100
S2 (serum)1U08.373.82
 1U58.506.44
 1U307.8925.0
 1U607.6530.9
 10U607.1130.6
 50U606.50100
S3 (serum)1U07.590.22
 1U57.889.45
 1U307.5112.7
 1U607.3919.6
 10U605.9557.1
 50U606.05100
S4 (serum)1U08.115.79
 1U58.395.76
 1U308.1112.4
 1U607.7728.6
 10U606.4799.6
 50U606.24100

Background Signal Control and Lower Limit of Detection.

Because HBV virion should only contain rcDNA, virions purified from 4 randomly selected serum samples were used as templates for cccDNA background signal measurement. As shown in Table 3, only total HBV DNA, but not cccDNA, was detectable with the Invader assay in all 4 samples. Two previously published PCR-based methods were also used as a comparison.20, 27 As determined by the presence or absence of corresponding bands on agarose gels, all samples had detectable total HBV DNA and undetectable cccDNA.

Table 3. Total HBV DNA and cccDNA Detection in Purified HBV Virion Sample by Three Different Methods
SamplesInvader Assays, log10 (copies/mL)PCR as in Mason et al.27PCR as in Stoll-Becker et al.20
  1. NOTE: +/− was determined by the presence/absence of bands in ethidium bromide stained gel.

  2. Abbreviation: UD, undetectable.

V1   
 Total HBV DNA4.59++
 cccDNAUD
V2   
 Total HBV DNA5.68++
 cccDNAUD
V3   
 Total HBV DNA5.53++
 cccDNAUD
V4   
 Total HBV DNA4.55++
 cccDNAUD

Using serially diluted plasmid standard as templates, the lower limit of detection of the Invader assay was determined to be 50 copies/assay, with a dynamic range of 5 orders of magnitude. When less than 50 copies was used, FOZ was less than 1 (data not shown). From this we calculated that the lower limit of detection was 0.002 copies/cell and 104 copies/mL for intrahepatic and serum HBV DNA, respectively.

Comparison Between Liver-Free and Total Liver Extracts.

To estimate the possible contribution of integrated HBV DNA to the signal detected by the Invader assay, the total HBV DNA and cccDNA content in liver-free and total liver DNA extracted was measured in 6 randomly selected samples. As shown in Table 4, the differences in both total HBV DNA and cccDNA between the total liver and liver-free DNA extract was less than 0.5 log, compatible with experimental variation.29, 30

Table 4. Total HBV DNA and cccDNA Detection in Total Liver and Liver-Free DNA Extracts
SamplesHBeAg Status Total Liver DNA Extract by QIAamp Method, log10 (copies/mL of extract)Liver-free DNA Extract by Modified Hirt Method, log10 (copies/mL of extract)Log Difference
B3HBeAg+Total HBV DNA8.158.370.21
  cccDNA7.037.270.24
B4HBeAg+Total HBV DNA5.786.030.25
  cccDNA6.026.380.25
B5Anti-HBe+Total HBV DNA6.236.530.31
  cccDNA6.236.530.31
B6Anti-HBe+Total HBV DNA6.026.380.36
  cccDNA5.345.540.20
B7Anti-HBe+Total HBV DNA8.668.560.10
  cccDNA7.397.070.32
B8Anti-HBe+Total HBV DNA7.447.660.22
  cccDNA7.447.660.22

Interassay and Intra-assay Variability.

The within-run coefficient of variation was determined for each individual Invader reaction in duplicates. The mean within-run percentage of coefficient of variation for the total HBV DNA detection (probe set MS1), cccDNA proportion detection (probe set PS2), and hgDNA detection (hgDNA probe set) were 4.5% (range: 0.04%–15.4%), 6.5% (range: 0.8%–18.4%), and 7.6% (range: 0.09%–26.9%), respectively. The results for the 4 interassay variation runs are shown in Table 5. The between-run percentages of coefficient of variation for the intrahepatic total HBV DNA and cccDNA detection were less than 12% and 23%, respectively.

Table 5. Interassay Variability of 6 Samples Using the Invader Assay for the Quantitation of Intrahepatic hgDNA, Total HBV DNA, and cccDNA
Sample No.Mean Human Genomic DNA/Assay ± SD (ng)Coefficient of VariationMean Total HBV DNA ± SD (copies/ng hgDNA)Coefficient of VariationMean cccDNA ± SD (copies/ng hgDNA)Coefficient of Variation
  1. NOTE: Data obtained from 4 independent assays on 4 consecutive days for each sample. Coefficient of variation = standard deviation/mean.

18.99 ± 0.323.59%767.91 ± 63.068.21%150.23 ± 23.9515.94%
24.94 ± 0.5010.09%1432.91 ± 66.634.65%163.05 ± 29.3017.97%
311.92 ± 1.8015.08%23562.12 ± 2243.269.52%1891.83 ± 222.0111.73%
41.565 ± 0.3019.28%75682.33 ± 8550.8511.30%3228.62 ± 715.4522.16%
52.553 ± 0.249.41%1032.47 ± 45.074.37%71.54 ± 12.2317.09%
62.60 ± 0.3111.73%1369.76 ± 151.5311.06%394.29 ± 86.7221.99%

Intrahepatic HBV DNA Detection.

The Invader assay was performed using DNA extract from liver biopsy samples. All HBeAg-positive patients and 33/36 (92%) of the anti–HBe-positive patients showed detectable intrahepatic total HBV DNA levels. All 5 non–hepatitis B control patients had undetectable HBV DNA levels. Of the patients with detectable intrahepatic total HBV DNA, 1 anti–HBe-positive patient had undetectable cccDNA levels.

Compared with HBeAg-positive patients, anti–HBe-positive patients had lower median total intrahepatic HBV DNA (12.8 [range: undetectable to 670] vs. 94.0 [range: 4.9–550] copies/cell, respectively; P < .001) and lower median intrahepatic cccDNA levels (0.75 [range: undetectable to 22.7] vs. 3.2 [range: 0.57–23.3] copies/cell, respectively; P = .001). However, there was no significant difference between the median intrahepatic cccDNA-to-total HBV DNA percentage in anti–HBe-positive and HBeAg-positive patients (5.5% [range: 0%–99.6%] vs. 4.7% [range: 1.97%–13.0%], respectively; P = .242).

Serum HBV DNA Detection.

Serum total HBV DNA levels were measured using both the COBAS-AM assay and the Invader assay. Forty-four out of 52 (85%) CHB patients had HBV DNA levels above the detection limit of the Invader assay. Among the 8 patients in whom HBV DNA was undetectable with the Invader assay, 1 also had HBV DNA that was undetectable with the COBAS-AM assay. For the 44 patients who had HBV DNA that was detectable with the Invader assay, the total serum HBV DNA levels in both assays had a very good correlation (r = 0.847, P < .001; Fig. 6A). Because the correlation was very good both at the upper and lower range of the Invader assay, and because the COBAS-AM assay could detect down to 200 copies/mL of serum HBV DNA, subsequent analyses involving serum HBV DNA levels were performed with the COBAS-AM assay data only. The anti–HBe-positive patients had lower serum HBV DNA levels than the HBeAg-positive patients (7.87 × 106 [range: <200–1,430 × 106] vs. 248 × 106 [range: 1.86 × 106–3,640 × 106] copies/mL respectively; P = .001).

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Figure 6. (A) Correlation between serum total HBV DNA measurement using the COBAS-AM assay and the Invader assay. (B) Relationship between the percentage of cccDNA/total HBV DNA and the amount of total HBV DNA in liver biopsy samples. HBV, hepatitis B virus; COBAS-AM assay, COBAS AMPLICOR HBV MONITOR Test; cccDNA, covalently closed circular DNA.

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The Invader assay also measured HBV cccDNA levels in sera. cccDNA was detectable in sera in 94% (15/16) of the HBeAg-positive patients and in 67% (24/36) of the anti–HBe-positive patients. Patients whose serum HBV DNA levels were lower than 7.53 × 105 copies/mL all had undetectable cccDNA in their sera. HBeAg-positive patients had higher median serum cccDNA levels compared with those of anti–HBe-positive patients (17.0 × 104 [range: undetecable to 1.09 × 107] vs. 2.87 × 104 [range: undetecable to 3.15 × 106] copies/mL, respectively; P = .002). There was no significant difference between the median serum cccDNA-to-total HBV DNA percentage in anti–HBe-positive and HBeAg-positive patients (3.50% [range: 0%–25%] vs. 3.54% [range: 0%–7.8%], respectively; P = .842).

Correlation of Intrahepatic and Serum Total and Covalently Closed Circular HBV DNA Levels.

Serum and intrahepatic total HBV DNA levels were found to correlate directly with each other (r = 0.778, P < .001). All patients who had intrahepatic total HBV DNA levels and intrahepatic cccDNA levels lower than 1.2 copies/cell and 0.12 copies/cell, respectively, had undetectable cccDNA in their sera. The serum cccDNA levels were also found to positively correlate with intrahepatic cccDNA levels (r = 0.481, P = .002) and serum total HBV DNA levels (r = 0.809, P < .001). Although the correlation between serum cccDNA and intrahepatic cccDNA was highly significant statistically, the correlation coefficient (r) was relatively low (0.481). There was also significant correlation between serum cccDNA and alanine aminotransferase levels, though the correlate coefficient was even lower (r = 0.278, P = .046).

There was a strong correlation between the cccDNA levels and total HBV DNA in the biopsy samples (r = 0.950, P < .001). However, it is found that when the total intrahepatic HBV DNA level was high, a large proportion was in the replicative rcDNA form. When the total intrahepatic HBV DNA level was low, the predominant form was nonreplicative cccDNA. Thus there was a strong negative correlation between the amount of total intrahepatic HBV DNA and the proportion of intrahepatic HBV DNA in the form of cccDNA (i.e., cccDNA/total intrahepatic HBV DNA × 100%) (r = −0.822, P < .001; Fig. 6B).

Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The Invader assay provides a specific method for the detection of both total HBV DNA and cccDNA. It detects only one strand of a DNA duplex.24, 31–33 The cccDNA probe set PS2 specifically detects the HBV PS sequences at the 5′ base of DR2, and successful hybridization would depend on the completion of the PS. Signal generation requires the simultaneous hybridization of the two oligonucleotides in set PS2 to the completed HBV target PS sequence to form a structure recognizable by Cleavase. With the specificity of both the enzyme and the two oligonucleotides, cccDNA can be detected specifically.

The DL and integrated forms of HBV DNA may also contain completed DNA sequences at the 5′ base of DR2. In DL DNA, the terminal redundancy (r) region, hence the 5′ base of DR2, is likely to be altered.9, 34 Although it is possible that a minute amount of unaltered r in DL DNA may generate a positive signal with the Invader probe set PS2, the DL form, like cccDNA, does not undergo active viral replication. Thus, the clinical significance of the DL form is similar to cccDNA. Although the exact viral sites of HBV DNA integration are variable, it often occurs at the two 11-bp DRs. This would lead to the breaching of the two regions so that the integrated HBV DNA would not be detected by the probes of the Invader assay.27, 35 Our observation that the total HBV DNA and cccDNA content in the liver-free and total liver DNA extracts were comparable further confirms that the contribution of integrated HBV DNA to the Invader assay signal is very small. Theoretically, the liver-free extraction method23 could be used for all our samples, but a commercial purification method was chosen for its convenience. Because the contribution of DL and integrated HBV DNA is likely to be minimal, the majority of the signal detected by the Invader probe set PS2 should originate from cccDNA.

This specificity of the Invader probe set PS2 to cccDNA was demonstrated using the synthetic oligonucleotide test. In the absence of a covalent linkage between the 5′ base of DR2 and the 3′ end of the PS of HBV DNA, even with all the nucleotides present, there was no detection of a cccDNA-specific signal by the Invader probe set PS2. The S1 nuclease experiment further confirmed the specificity of the Invader assay for cccDNA in liver tissues and serum samples. The treated samples showed progressively higher proportion of cccDNA with an increasing incubation time and quantity of S1 nuclease used, the most robust of which (50-U, 60-minute incubation) led to a homogenous population of 100% of cccDNA.

cccDNA was not detectable in purified HBV virions by both the Invader assay and two previously published PCR methods. This indicates that the background signal of the Invader assay was minimal when detecting a homologous population of rcDNA. These results correspond to the selective PCR methods previously used.

Interassay and intra-assay variation data showed that the Invader assay has a good reproducibility. The serum total HBV DNA levels obtained by the Invader assay had a very good correlation to the levels obtained with the COBAS-AM assay. However, it can be noted that overall there was an approximately tenfold difference between the total HBV DNA data obtained with the COBAS-AM assay and the Invader assay. This is partly due to the difference in the extraction efficiency between the QIAamp DNA extraction kit and the direct lysis method used in the COBAS-AM assay. The possibility that the COBAS-AM assay may be more sensitive than the Invader assay cannot be excluded. Nevertheless, the overall result indicated that the Invader assay is reliable for the measurement of serum viral load.

To our knowledge, this is the first study that quantifies HBV cccDNA in serum, although it has been reported qualitatively that extracellular cccDNA is detected in medium of cultured HepG2.2.15 cells36 and in serum of HBV-infected immunocompetent rats with transplanted human hepatocytes.37 The source of HBV cccDNA in serum is uncertain. It is possible that serum cccDNA originated from the lysis of infected hepatocytes. The release of naked HBV nucleic acid into the bloodstream has been reported previously.38–40 However, the correlation coefficient between serum and intrahepatic HBV cccDNA was relatively low at 0.481. Moreover, serum cccDNA correlated only weakly with alanine aminotransferase levels, which reflect the degree of hepatocyte lysis. This may be due to the uneven distribution of cccDNA in the liver,41 which would mean that liver biopsy specimens may not represent the overall intrahepatic cccDNA content. The relatively low correlation coefficients may also indicate that lysed hepatocytes may not be the exclusive source of serum cccDNA. Previous reports have demonstrated the presence of cccDNA in peripheral blood mononuclear cells,20, 42, 43 which could be another source of cccDNA in the serum. However, it is uncertain whether or not the extrahepatic pool of HBV cccDNA in peripheral mononuclear cells can replicate, and the clinical significance of its presence has yet to be determined.

This study showed that both intrahepatic and serum cccDNA levels were more dynamic than expected. Intrahepatic cccDNA was found to correlate positively with the total intrahepatic HBV DNA. The observation that the proportion of intrahepatic covalently closed circular HBV DNA increased as the amount of total intrahepatic HBV DNA decreased showed that cccDNA became the dominant form of intrahepatic HBV DNA as the viral replication became less active. Thus, even at a late stage of CHB in the Chinese, there would still be a stable pool of intrahepatic HBV DNA, which may explain the frequent viral exacerbation observed in anti–HBe-positive patients.44, 45 Another study of 92 patients with HBsAg seroclearance found that although 62% of patients had undetectable intrahepatic HBV DNA, in the remaining 38% of patients the majority (70%–100%) of intrahepatic HBV DNA is in the form of cccDNA (median levels: 0.006 copies/cell).46 The presence of cccDNA in late-stage CHB has obvious implications for antiviral therapy with nucleoside analogues. Currently, it is advocated that nucleoside analogues should be stopped after stable HBeAg seroconversion with HBV DNA levels dropping below 105 copies/mL.47, 48 However, there is evidence that (at least in Chinese CHB patients) cirrhosis continues to progress, and complication of cirrhosis can occur with HBV DNA levels below 105 copies/mL.49 The presence of a significant amount of cccDNA at this stage of the disease may provide the source for continuing viral activity.

In conclusion, HBeAg-positive patients had higher serum and intrahepatic total HBV DNA and cccDNA levels than anti–HBe-positive patients. As the total viral load decreased, cccDNA became the predominant form of intrahepatic HBV DNA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Tamara Sander from Third Wave Technologies, Inc., and Lynn Condreay and Eric Bourne from GlaxoSmithKline for their excellent technical support in the Invader assay. We also thank Charles Cheng, Vincent Ngai, Ringo Wu, John Young, and John Yuen for their dedicated support in clinical laboratory work and Ting Kin Cheung for his helpful suggestion.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References