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Abstract

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

Although threshold levels for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) titers have recently been proposed to guide therapy for chronic hepatitis B (CHB), their relationship to circulating hepatitis B virus (HBV) DNA and intrahepatic HBV replicative intermediates, and the significance of emerging viral variants, remains unclear. We therefore tested the hypothesis that HBsAg and HBeAg titers may vary independently of viral replication in vivo. In all, 149 treatment-naïve CHB patients were recruited (HBeAg-positive, n = 71; HBeAg-negative, n = 78). Quantification of HBeAg and HBsAg was performed by enzyme immunoassay. Virological characterization included serum HBV DNA load, HBV genotype, basal core promoter (BCP)/precore (PC) sequence, and, in a subset (n = 44), measurement of intrahepatic covalently closed circular DNA (cccDNA) and total HBV DNA, as well as quantitative immunohistochemical (IHC) staining for HBsAg. In HBeAg-positive CHB, HBsAg was positively correlated with serum HBV DNA and intrahepatic cccDNA and total HBV DNA (r = 0.69, 0.71, 0.76, P < 0.01). HBeAg correlated with serum HBV DNA (r = 0.60, P < 0.0001), although emerging BCP/PC variants reduced HBeAg titer independent of viral replication. In HBeAg-negative CHB, HBsAg correlated poorly with serum HBV DNA (r = 0.28, P = 0.01) and did not correlate with intrahepatic cccDNA nor total HBV DNA. Quantitative IHC for hepatocyte HBsAg confirmed a relationship with viral replication only in HBeAg-positive patients. Conclusion: The correlation between quantitative HBsAg titer and serum and intrahepatic markers of HBV replication differs between patients with HBeAg-positive and HBeAg-negative CHB. HBeAg titers may fall independent of viral replication as HBeAg-defective variants emerge prior to HBeAg seroconversion. These findings provide new insights into viral pathogenesis and have practical implications for the use of quantitative serology as a clinical biomarker. (HEPATOLOGY 2010)

Recent developments in the treatment of chronic hepatitis B (CHB) have led to interest in novel biomarkers for predicting treatment response. Quantitative serology for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) are two promising candidates. Preliminary data have supported the clinical utility in treatment protocols for pegylated-interferon α (PEGIFN) and nucleos(t)ide analogs (NA), respectively.1, 2

HBsAg seroconversion is the ultimate laboratory marker of successful therapy for patients with CHB. Data from clinical trials suggest a role for quantification of HBsAg at baseline and/or on-treatment in identifying patients most likely to achieve HBsAg seroconversion.3, 4 Mathematical modeling of HBsAg decline on treatment has also been used to predict the duration of therapy required for HBsAg seroconversion for both IFN- and NA-based therapies.5 There are a number of assays that have been developed to measure quantitative HBsAg titer.5-8 Importantly, these tests are inexpensive (<10% of the cost of a serum hepatitis B virus [HBV] DNA assay) and use high-throughput platforms.

HBeAg seroconversion is an established therapeutic endpoint for the management of HBeAg-positive CHB and is associated with reduced morbidity and mortality.9 Quantification of HBeAg has recently been evaluated as a biomarker during PEGIFN therapy.1 In a large study of patients with HBeAg-positive CHB, a baseline HBeAg titer of ≤31 Paul Ehrlich (PE) IU/mL was associated with a positive predictive value of 54% for HBeAg seroconversion following 48 weeks of therapy. Furthermore, a failure of HBeAg titer to decline to <100 PE IU/mL after 24 weeks of therapy was associated with a negative predictive value for HBeAg seroconversion of 96%, greater than that obtained from monitoring levels of serum HBV DNA. Quantitative enzyme immunoassays (EIAs) for HBeAg are not widely commercially licensed, and this has contributed to these assays remaining research tools.

HBeAg and HBsAg titer have both been proposed as surrogates for infected liver cell mass, or HBV covalently closed circular DNA (cccDNA), the hepatocyte nuclear reservoir that is responsible for viral persistence.10 This concept underpins their use as biomarkers. If the hepatocyte is considered in isolation, HBsAg, HBeAg, and serum HBV DNA would be expected to directly correlate with each other, and with liver cccDNA, as all are translated from separate transcripts (Pre-S1, Pre-S2/S, precore/core, and pregenomic messenger RNA [mRNA], respectively) directly derived from cccDNA. However, the published data that describes these relationships are limited and conflicting. Positive correlations have been observed between HBeAg or HBsAg titer and serum HBV DNA in some studies,6 but not all.2, 5, 11 Similarly, the positive correlation between HBsAg titer and HBV cccDNA/total intrahepatic (IH) HBV DNA that has previously been described,8, 12 was not seen in two recent studies.13, 14 Importantly, most of the studies of HBsAg and HBV cccDNA involved small cohorts and were limited by the sensitivity of the available assays for serum HBV DNA. In addition, studies examining HBsAg quantification have not compared HBeAg-positive and HBeAg-negative populations, known to be characterized by distinct immunological milieu and different levels of virion productivity.12, 15 Studies of HBeAg have not previously considered the emergence of A1726T/G1764A basal core promoter (BCP) or G1896A precore (PC) variants coexisting with wildtype (WT) HBV in the viral quasispecies pool. Such variants are known to emerge in the years leading up to HBeAg seroconversion.16 Although the BCP and the PC variants have been shown to reduce or abolish HBeAg production, respectively, in vitro,17 their impact on HBeAg titer in vivo, in the context of the viral quasispecies pool and an active immune system, has not been defined. The relationship between HBeAg titer and IH markers of HBV has not been investigated.

Here we provide the first detailed and comprehensive analysis of serum HBsAg and HBeAg titers within a treatment-naïve patient cohort, with a focus on their relationship to serum and liver markers of HBV replication, as well as determining the significance of emerging viral variants. We tested the hypothesis that HBsAg and HBeAg titers may vary independently of viral replication in vivo.

Patients and Methods

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

Patients

The patient cohort was collected from three separate clinical sites: 1) St. Vincent's Hospital, Melbourne, Australia (n = 110)18; 2) Auckland City Hospital, Auckland, New Zealand (n = 15); and 3) Queen Mary Hospital, University of Hong Kong, Hong Kong Special Administrative Region of China (n = 30). All patients were positive for HBsAg for at least 6 months prior to enrolment and had detectable HBV DNA in the serum. All HBeAg-positive patients had documented elevation of serum alanine aminotransferase (ALT) for 3-6 months prior to liver biopsy (ALT >30 IU/mL for men, and >19 IU/mL for women19). No patients had evidence of decompensated liver disease or hepatocellular carcinoma. All patients tested negative for hepatitis C virus (HCV), hepatitis D virus, and human immunodeficiency virus (HIV); markers for coexistent autoimmune or metabolic liver disease were negative. Liver biopsy for routine histology was available in 73 patients from the Australian center; all results were reported using the METAVIR scoring system.20 Fresh-frozen liver tissue was collected for analysis of intrahepatic HBV replicative intermediates in a subset of patients from the Australian site (n = 44/73) (see below). Serum samples for biochemistry and HBV virological studies were collected on the same day as liver biopsy. Institutional ethics approval for the research protocol was obtained and all patients provided informed consent according to the Declaration of Helsinki.

Quantitative HBsAg Assay

Serum HBsAg titer was measured by EIA using the ARCHITECT platform (Abbott Laboratories, Chicago, IL), as per the manufacturer's instructions, which expresses HBsAg against an internal World Health Organization reference standard in IU/mL.

Quantitative HBeAg Assay

The development of the quantitative HBeAg assay was based on the method of Perrillo et al.21 Serum HBeAg was measured by EIA (ARCHITECT platform; Abbott Laboratories). Serial dilutions of the PE HBeAg reference standard (Paul-Ehrlich Institute, Langen, Germany) were used to define the linear range of the assay and generate a reference curve for linear regression (described in detail in Supporting Methods).

HBV Viral Load Analysis

Patients recruited from St. Vincent's Hospital and Auckland Hospital had HBV viral load testing performed using the Versant HBV DNA 3.0 assay (bDNA) (Siemens Healthcare Diagnostics, Tarrytown, NY) according to the manufacturer's instructions. Patients recruited from the Queen Mary Hospital in Hong Kong were tested using the Digene Hybrid capture II assay with the ultrasensitive detection protocol (Digene Diagnostics, Beltsville, MD). The lower limit of detection (LLD) for both assays is ≈102-3 IU/mL. The Digene assay viral load results were converted to the international reference unit for analysis by approximating 1 pg = 280,000 copies and 1 IU/mL = 5.7 copies/mL, allowing comparison between the assays.

Serum HBV DNA Polymerase Chain Reaction (PCR) and Sequence Analysis

Specimen Extraction.

HBV DNA was extracted from 200 μL of patient serum using the QIAamp DNA MiniKit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA was eluted in a final volume of 50 μL of supplied elution buffer.

HBV DNA Amplification and Sequence Analysis.

Two different PCR reactions were used to amplify the polymerase and BCP/PC regions of the HBV genome, as previously described.22 HBV genes were analyzed using the online HBV genome analysis program (SeqHepB: www.seqvirology.com). This program compares input sequence data with known reference sequences to determine HBV genotype, variants, and mutations associated with antiviral resistance.23 The PC variant was defined by the presence of the G1896A mutation (PC mutation) and the BCP variant by the A1762T/G1764A mutation (BCP mutation).

Quantification of HBV cccDNA and Total HBV DNA from Liver Tissue by Real-Time PCR

Intrahepatic HBV cccDNA and total IH HBV DNA levels were quantified by real-time PCR (RT-PCR) using a LightCycler instrument (Roche Diagnostics, Mannheim, Germany) as described.24 β-Globin was used as the housekeeping gene (LightCycler Control Kit DNA, Roche Diagnostics) to allow standardization of the extracted DNA and expression of HBV cccDNA and total IH HBV DNA as copies per genome equivalent (c/Geq).

Quantitative Immunohistochemistry (IHC) for HBsAg

Immunostaining of HBV antigens was performed on sections from paraffin-embedded liver biopsies. The primary antibody used was a mouse monoclonal against HBsAg (M3506, Dako, Carpinteria, CA). The chromogen used was diaminobenzidine (DAB) and staining was performed using BenchMark XT automated staining system (Ventana, Tucson, AZ). Liver sections were scored by microscopic examination by two investigators (D.I., J.S.) blinded to clinical data, using a semiquantitative scale of 0-4, corresponding to positivity in 0, <5, 5-9, 10-29, ≥30% of hepatocytes, respectively (modified from Ref.25). Intensity of staining was judged semiquantitatively as absent, minimal, moderate, or strong.25 Staining pattern was reported as cytoplasmic, membranous, or absent, as described.26, 27

Statistical Analysis

Continuous and categorical variables were compared between groups using, respectively, the Mann-Whitney test and the chi-square/Fisher's exact test. Pearson's correlation coefficient (r) was used to describe the correlation between two continuous, normally distributed variables. A Spearman's correlation was used where variables were not normally distributed. All statistical analyses were performed using SAS v. 9.1 (SAS Institute, Cary, NC).

Results

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

One hundred and forty-nine treatment-naïve patients with CHB were recruited into the study (Table 1). The majority of patients were Asian and infected with genotype B or C HBV. All 71 HBeAg-positive patients were in the immuno-elimination phase of disease, defined by persistently elevated serum ALT. Of the HBeAg-negative patients, 57/78 (73%) were in the reactivation phase of disease, defined by serum HBV DNA persistently >2,000 IU/mL and raised ALT.28 Twenty-one (27%) were in the low replicative phase of disease (serum HBV DNA <2,000 IU/mL). Patients with HBeAg-positive CHB were younger, had higher serum ALT and HBV DNA levels, and were more likely to be infected with genotype C HBV. In patients with HBeAg-negative CHB, median ALT was higher in patients with viral load >2,000 IU/mL (median 54 [25th-25th centile 38-84] versus 25 [22-36], P < 0.0001). Liver histology was available in 73 patients (HBeAg-positive n = 22, HBeAg-negative n = 51). More patients with HBeAg-negative CHB had METAVIR F3-4 fibrosis. In the subset of patients in whom IH HBV replicative markers were measured, patients with HBeAg-positive CHB had higher levels of HBV cccDNA and total IH DNA.

Table 1. Baseline Patient Data
  OverallHBeAg+HBeAg−P Value
  1. Statistical comparison between HBeAg-positive and HBeAg-negative patients was performed using the Mann-Whitney U test (MWUT) for continuous data and the chi-squared test for categorical data. c/Geq = copies per genome equivalent.

N 1497178 
Age (years)Median403348<.0001
 25th-75th centile28-4826-4037-52 
AsianN13363700.8422
 %89%89%90% 
MN9642540.1994
 %64%59%69% 
ALT (IU/mL)Median6510745<.0001
 25th-75th centile38-12465-17828-71 
Viral loadMedian6.27.34.6<.0001
(log10 IU/mL)25th-75th centile4.4-7.56.8-8.33.2-5.8 
Viral load <2000 IU/mLN21021<.0001
 %14%0%27% 
METAVIR F3-4N13/732/2211/510.32
 %18%9%22% 
Genotype     
 A 1%2%1%1.0000
 B 47%35%58%0.0073
 C 40%58%24%<.0001
 D 11%6%15%0.1019
 G 1%0%1%1.0000
Intrahepatic markers (n = 44)(n = 17)(n = 27) 
cccDNA (c/Geq)Median0.433.650.2<0.0001
 25th-75th centile0.15-2.301.43-8.550.10-0.43 
Total IH HBV DNA (c/Geq)Median3.09207.661.19<0.0001
 25th-75th centile0.64-204.7139.17-420.960.40-3.09 

Distribution of HBsAg Titers.

The distribution of serum HBsAg titer across the study population is illustrated in Fig. 1A,B. The distribution was skewed (median 3,940 IU/mL, range 3-794,000 IU/mL). The median HBsAg titer was significantly higher in patients with HBeAg-positive CHB compared to HBeAg-negative CHB (median 9,459 versus 1,642 IU/mL, P < 0.0001) (Fig. 1C). Interestingly, when HBsAg was adjusted for viral load the relationship was reversed, suggesting that HBsAg production was relatively preserved at the lower viral load “set-point” observed in HBeAg-negative CHB (Fig. 1D). We did not identify any HBsAg thresholds that could meaningfully differentiate patients according to viral load strata, in contrast to a previous report.29 There was no statistical association between serum ALT levels and serum HBsAg titer (Table 2). Serum HBsAg titers were also analyzed according to age, gender, viral genotype, and METAVIR fibrosis score. None of these variables were associated with HBsAg titer after correction for viral load and HBeAg status (data not shown).

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Figure 1. (A) Population distribution of HBsAg titer (n = 149), comparing patients with HBeAg-positive versus HBeAg-negative CHB in the overall cohort. (B) Comparison for patients with HBsAg titers <10,000 IU/mL (n = 99). (C) Plot of HBsAg titer (log IU/mL) according to HBeAg status (n = 149); P < 0.0001, Mann-Whitney U test. (D) Plot of HBsAg titer (log IU/mL) adjusted for viral load, according to HBeAg status; P < 0.0001, Mann-Whitney U test.

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Table 2. Correlation Between HBsAg and HBeAg Titer and Serum and Intrahepatic HBV Replicative Markers
    R*P
  • *

    Correlation coefficient for comparisons of logarithmically transformed data.

HBeAg-positive patients     
N = 71HBsAgSerum HBV DNA 0.6863<.0001
  ALT 0.069890.5654
  cccDNA(N = 17)0.71100.0014
  total IH HBV DNA(N = 17)0.758150.0004
 Serum HBV DNAALT 0.08720.4728
  cccDNA(N = 17)0.80997<.0001
  Total IH HBV DNA(N = 17)0.97471<.0001
 HBeAgSerum HBV DNA 0.6023<.0001
  HBsAg 0.5947<.0001
  ALT 0.01420.9071
  cccDNA(N = 17)0.40010.1115
  Total IH HBV DNA(N = 17)0.59900.0111
HBeAg-negative patients     
OverallHBsAgSerum HBV DNA 0.28180.0124
N = 78 ALT 0.15220.1836
  cccDNA(N = 27)0.15040.4539
  Total IH HBV DNA(N = 27)0.25250.2038
 Serum HBV DNAALT 0.4591<.0001
  cccDNA(N = 27)0.29790.1312
  Total IH HBV DNA(N = 27)0.8371<.0001
Low replicativeHBsAgSerum HBV DNA 0.14640.5267
Serum HBV DNA < 2,000 IU/mL ALT −0.11250.6272
N = 21 cccDNA(N = 1)**
  Total IH HBV DNA(N = 1)**
ReactivationHBsAgSerum HBV DNA 0.31720.0162
Serum HBV DNA > 2,000 IU/mL ALT 0.16820.2112
N = 57 cccDNA(N = 26)0.14740.4725
  Total IH HBV DNA(N = 26)0.25090.2164

Correlation of HBsAg with Markers of HBV Replication.

HBsAg titer was positively correlated with serum HBV DNA (r = 0.58, P < 0.0001) and HBeAg (r = 0.60, P < 0.0001) (Fig. 2; Table 2). The correlation between HBsAg titer and serum HBV DNA was stronger in patients with HBeAg-positive CHB (r = 0.69, P < 0.0001). In contrast, the correlation between HBsAg-titer and viral load were weakly correlated in the overall HBeAg-negative cohort (r = 0.28, P = 0.012). Among HBeAg-negative patients in the inactive phase (<2,000 IU/mL), HBsAg titer did not correlate with HBV DNA (r = 0.15, P = 0.53).

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Figure 2. (A) Correlation of serum HBsAg titer and HBV DNA level, according to HBeAg status (HBeAg-positive, r = 0.69, P < 0.0001, HBeAg-negative, r = 0.28, P = 0.01). (B) Correlation of serum HBsAg titer and total IH HBV DNA level, according to HBeAg status (HBeAg-positive, r = 0.76, P = 0.0004, HBeAg-negative, r = 0.25, P = 0.20). (C) Correlation of serum HBsAg titer and HBV cccDNA level according to HBeAg status (HBeAg-positive, r = 0.71, P= 0.001, HBeAg-negative, r = 0.15, P = 0.45). Data were not normally distributed and were therefore log transformed.

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In 44 patients (17 HBeAg-positive and 27 HBeAg-negative), serum HBsAg titer was then correlated with the IH markers of HBV replication (the relationship between HBsAg titer and serum HBV DNA in the smaller subset was similar to that in the larger cohort, r = 0.63, P < 0.001). Again, HBsAg titer was also only correlated with IH replicative intermediates in the HBeAg-positive patients, and not in the HBeAg-negative patients (Fig. 2; Table 2).

Quantitative IHC for HBsAg.

In these 44 patients we also performed quantitative immunohistochemistry for HBsAg on liver specimens (Fig. 3; Table 3). Specifically, we judged the liver specimens according to the percentage of hepatocytes staining positive (Score), the intensity of the scoring (Intensity) as well as the cellular distribution of staining (Pattern = no staining, cytoplasmic or membranous for HBsAg26, 27). In HBeAg-positive and HBeAg-negative patients, serum HBsAg titer was positively correlated with the number and intensity of HBsAg-positive hepatocytes (r = 0.50, P = 0.04, and r = 0.61, P = 0.001, respectively, Table 3). High serum HBsAg titers were associated with a membranous staining pattern (P < 0.001; Fig. 3).

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Figure 3. In patients with HBeAg-negative CHB, hepatocyte HBsAg expression, as measured by IHC, may remain relatively preserved despite low-level viral replication. The plots present correlation of (A) serum HBV DNA level and HBsAg score (percent hepatocyte staining) according to HBeAg status (HBeAg-positive, r = 0.43, P = 0.08, HBeAg-negative, r = 0.05, P = 0.77). (B) Total IH HBV DNA level and HBsAg score (percent hepatocyte staining) according to HBeAg status (HBeAg-positive, r = 0.45, P = 0.07, HBeAg-negative, r = 0.32, P = 0.10). (C) Serum HBV DNA level and HBsAg intensity according to HBeAg status (HBeAg-positive, r = 0.54, P = 0.02, HBeAg-negative, r = 0.01, P = 0.96). (D) Total IH HBV DNA level and HBsAg intensity according to HBeAg status (HBeAg-positive, r = 0.53, P = 0.03, HBeAg-negative, r = 0.27, P = 0.17). (E,F) Comparisons of serum HBV DNA level and total IH HBV DNA according to the pattern of hepatocyte HBsAg staining according to HBeAg status.

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Table 3. (A) Correlation Between HBsAg, HBeAg Titer, Serum/Intrahepatic HBV Replicative Markers, and Quantitative IHC Staining of Liver Biopsies for HBsAg. (B) Kruskal-Wallis Tests for Trend in HBsAg Titer, HBeAg Titer, Serum HBV DNA, cccDNA, and Total IH DNA According to Staining Pattern for HBsAg in Patients with HBeAg-Positive and HBeAg-Negative CHB, Respectively
Quantitative ImmunohistochemistrySpearman R*P
A.    
HBeAg-positiveHBsAgS-score0.49360.0440
N = 17 S- intensity0.60620.0099
 HBeAgS-score0.57340.016
  S- intensity0.58930.0128
 Serum HBV DNAS-score0.43160.0837
  S- intensity0.54110.0249
 cccDNAS-score0.16830.5184
  S- intensity0.28290.2712
 Total IH HBV DNAS-score0.45060.0695
  S- intensity0.52930.0289
HBeAg-negativeHBsAgS-score0.53940.0037
N = 27 S- intensity0.43920.0219
 Serum HBV DNAS-score0.05830.7726
  S- intensity0.01070.9577
 cccDNAS-score−0.08250.6825
  S- intensity−0.22060.2689
 Total IH HBV DNAS-score0.32350.0997
  S- intensity0.27230.1695
 P
Qualitative Immunohistochemistry, S-patternK - W test
  1. S/C score = 0, no hepatocytes stained positive for HBsAg/HBcAg; 1, <5% hepatocytes; 2-5, 9% hepatocytes; 3-10, 29% hepatocytes; 4, >30% hepatocytes. S/C intensity = 0, none; 1, mild; 2, moderate; 3, strong.

  2. S-pattern = no staining, cytoplasmic or membranous staining.

B.  
HBeAg-positiveHBsAg0.0464
N = 17HBeAg0.0212
 Serum HBV DNA0.06
 cccDNA0.589
 Total IH HBV DNA0.0725
HBeAg-negativeHBsAg0.0147
N = 27Serum HBV DNA0.8836
 cccDNA0.1877
 Total IH HBV DNA0.2831

In patients with HBeAg-positive CHB, serum HBV DNA and total IH HBV DNA were positively correlated with the Intensity of HBsAg staining and a strong trend was noted for a positive correlation with the HBsAg Score (Fig. 3; Table 3). HBeAg positivity and high level viral replication were associated with a membranous pattern of HBsAg staining (Fig. 3). A negative correlation was noted between serum ALT and both hepatocyte HBsAg Score and Intensity (r = −0.53, P = 0.029, and 0.51, P = 0.036, respectively).

In contrast, no correlation was observed between viral replication (serum HBV DNA/total IH HBV DNA) and hepatocyte HBsAg Score/Intensity in patients with HBeAg-negative CHB. The pattern of staining was also distinct, with no patient having a membranous pattern of HBsAg (Table 3; Fig. 3). Of note, in two patients with serum HBV DNA levels <5,000 IU/mL, more than 30% of hepatocytes displayed HBsAg staining of at least moderate intensity. No relationship between serum ALT and HBsAg IHC was noted in HBeAg-negative patients.

Distribution of HBeAg Titers.

Seventy-one patients were HBeAg-positive (Table 1). The distribution of serum HBeAg titer is illustrated in Fig. 4A,B. The distribution was skewed (median 1,147 PE IU/mL, range = 1.5-14,236 PE IU/mL), with 35/71 (49%) patients having HBeAg titer <1000 PE IU/mL, and 13/71 (18%) an HBeAg titer of ≤31 PE IU/mL.1

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Figure 4. Population distribution of HBeAg titer (n = 71), for (A) the overall cohort and (B) patients with HBeAg titer <1000 PE IU/mL at right (n = 35) (ii). The maximum titer observed was 14,236 PE IU/mL. (C) Plot of HBeAg titer according to BCP/PC sequence, comparing patients with WT/WT sequence to patients with BCP and/or PC variant sequence (n = 61; in 10 patients BCP/PC sequence was not available); P < 0.0001, Mann-Whitney U test. (D) Plot of HBeAg titer adjusted for viral load, comparing patients with WT/WT sequence to patients with BCP and/or PC variant sequence; P < 0.0001, Mann-Whitney U test.

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Correlation of HBeAg with Markers of HBV Replication.

HBeAg titer correlated positively with HBV DNA (r = 0.60, P < 0.0001) (Table 2). In a subset (n = 17), serum HBeAg titer was also correlated with the IH markers of HBV replication. HBeAg correlated with total IH DNA (r = 0.60, P = 0.01) but not HBV cccDNA was (r = 0.40, P = 0.11) (Table 2). The relationship between HBeAg and HBV DNA in this subset (n = 17) was significant and identical to the larger cohort (r = 0.64, P = 0.005).

Analysis of the Impact of BCP/PC Variants on HBeAg Titer.

Sequencing of the BCP/PC region of the HBV genome was performed in 61 HBeAg-positive patients (sequencing was not possible in 10 patients for technical reasons). In 27 patients HBV DNA sequence was WT at both the BCP and PC sites, in 22 patients the BCP mutation alone was present, and in 10 patients PC mutation alone. Both were detected in two patients. Median HBeAg titer was higher in the WT group compared to the patients with either BCP or PC variants (3675 versus 80 PE IU/mL, P < 0.001) (Fig. 4C). It was possible that the lower HBeAg titer was secondary to reduced viral replication. However, the effect of the BCP/PC variants persisted after adjustment for viral load (Fig. 4D). A nonsignificant trend was also noted for lower HBeAg titer in the setting of PC variants compared to BCP variants, although this analysis was limited by the small numbers of HBeAg-positive patients with the PC variant as the dominant virus (data not shown). Serum HBeAg titers did not differ according to gender, serum ALT levels, viral genotype (B versus C), or METAVIR fibrosis score (data not shown).

Correlation of Serum HBV DNA with IH Markers of HBV Replication.

The relationship of serum HBV DNA with the IH markers of replication was analyzed (n = 44). This relationship was found to be strong (serum HBV DNA versus cccDNA, r = 0.75, P < 0.001, and serum HBV DNA versus total IH HBV DNA, r = 0.94, P < 0.0001), as reported.7, 15 Correlation of serum HBV DNA with total IH DNA was significant in both HBeAg-positive and HBeAg-negative CHB (r = 0.97, P < 0.0001, versus r = 0.84, P < 0.0001). However, serum HBV DNA correlated significantly with cccDNA only in HBeAg-positive patients (r = 0.81, P < 0.001, versus r = 0.30, P = 0.13 in HBeAg-negative CHB).

Discussion

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

This study provides the first detailed description of serum HBsAg and HBeAg titers across a large cohort of treatment-naïve patients with CHB. Both HBV antigens have recently been proposed as biomarkers for treatment response, as well as clinical surrogates for IH HBV cccDNA levels, the intranuclear HBV reservoir responsible for persistence. However, our data indicate that the relationships between HBsAg, HBeAg, serum HBV DNA, and HBV replicative intermediates are complex. HBsAg titer correlated with serum HBV DNA, IH HBV cccDNA, and total IH DNA only in patients with HBeAg-positive CHB, but not in patients with HBeAg-negative CHB, where HBsAg titers were preserved relative to HBV replication. HBeAg correlated with viral load, but emerging BCP/PC variants were observed to independently reduce HBeAg titer. Clinical interpretation of HBsAg and HBeAg titers might therefore be refined by considering the phase of disease, as well as quasispecies diversity.16

IHC studies showed for the first time that serum HBsAg titers correlated with the number and intensity of HBsAg-positive cells. However, the number and intensity of hepatocytes staining positive for HBsAg, as well as the cellular pattern of distribution, were related to HBV replication only in patients with HBeAg-positive CHB, confirming that the association between HBsAg production and HBV replication breaks down in the HBeAg-negative phase of disease. This might occur if HBsAg was produced from a source other than the intranuclear cccDNA (Fig. 5). In HBV infection, viral integration into the host genome has been observed to begin early in infection.30, 31 The observation that the number and intensity of HBsAg-positive cells in the liver may be preserved despite low-level HBV replication is consistent with the occurrence of integration. Although integration is believed to be a random event, a high preference for integration has been observed at the DR1 and DR2 sequences on the HBV genome,32 and sequences of the S genes are often present in integrated segments.33 Although integrated sequences cannot provide a template for productive viral replication, HBsAg may be produced.33 Progressive integration might therefore provide a template for persistent HBsAg production independent of viral load. To date, no method for reliably differentiating truncated HBs peptides in serum has been developed to test this hypothesis. Another explanation might involve preferential control of the viral replication pathway at the posttranscriptional level, sparing HBV cccDNA and HBsAg transcription/secretion. This has been demonstrated to occur in vitro in the setting of cytokine effects (targeting the encapsidation step,34), and is consistent with the more profound immune pressure that is present in the HBeAg-negative phase of disease. These two hypotheses are not mutually exclusive.

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Figure 5. Diagram of the lifecycle of HBV, highlighting the separate intracellular pathways for viral replication, HBsAg production and HBeAg production. HBeAg production may be reduced independent of viral load by the emergence of viral variants defective for HBeAg production (e.g., the G1896A PC variant). HBsAg production from integrated sequence may preserve titers independent of viral load. Immune-mediated inhibition of HBV replication (e.g., at the level of encapsidation) may selectively decrease HBV virion production. PC RNA = precore mRNA, pgRNA = pregenomic RNA.

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Serum ALT levels did not correlate with HBsAg titer in patients, regardless of HBeAg status. In contrast, a negative correlation was noted between serum ALT and the number and intensity of HBsAg-positive hepatocytes by IHC in the HBeAg-positive cohort. In this situation it is likely that the serum ALT level is a marker for more active anti-HBV immunity, suppressing hepatocyte HBV replication and capsid assembly. The fact that a similar correlation is not seen with serum HBsAg titer may reflect that subviral particles are produced far in excess of HBV capsids, confounding the relationship in the peripheral circulation. In HBeAg-negative disease, where serum ALT levels tend to be uniformly lower, and HBsAg expression may be independent of viral replication, no association was observed.

The correlation between HBeAg and HBV replicative markers was modest and the HBeAg titer did not correlate with liver HBV cccDNA (r = 0.40, P = 0.11). The data therefore implied another “disconnect,” this time between the HBeAg production pathway and the viral replication pathway (Fig. 5). The most likely explanation was that viral variants defective for HBeAg production were emerging before seroconversion, as we observed.16 The BCP/PC variants are the most common to be associated with HBeAg-negative CHB. The mechanisms of reduced or abolished HBeAg production have been defined in vitro. The ntA1762T and G1764A BCP mutations are thought to alter the binding of transcriptional proteins to reduce CP-directed transcription, decreasing pre-C mRNA levels and causing a relative reduction in HBeAg.35 The G1896A PC mutation encodes for a stop codon that prevents translation of the PC protein from PC mRNA.36 The emergence of these variants would therefore be expected to be associated with a decline in HBeAg titer independent of viral replication.

In this context, failure to consider quasispecies composition is the most likely explanation for previous conflicting descriptions of correlations between HBeAg and serum HBV DNA. Sequence analysis of the HBV quasispecies therefore should be considered when developing clinical algorithms that include HBeAg titer. That this might improve predictive power is an attractive hypothesis that requires further testing. In the setting of a homogenous WT viral population, a titer of ≤31 PE IU/mL may reflect an active immune system that will be responsive to antiviral therapy, particularly with an immune modulator such as PEGIFN. Such a group might achieve seroconversion rates of >50%. In contrast, an HBeAg titer of ≤31 PE IU/mL in the setting of emerging PC variant virus, and immune escape, may indicate a more treatment-refractory group.

In conclusion, quantitative HBeAg and HBsAg assays are inexpensive, can be used to screen large numbers of samples, and are compatible with automation. It is clear that they can offer much more relevant data than can be obtained by standard qualitative serology. They have the potential to aid decisions regarding treatment initiation and may open the way for a response-guided therapy approach in CHB. However, the relationship between HBsAg and HBeAg titers, IH replicative intermediates, and serum HBV DNA is complex, and is likely to reflect an interplay between virological and host immunological factors. We have observed key differences in HBsAg expression comparing patients with HBeAg-positive and HBeAg-negative CHB. An important role for the emergence of BCP/PC variant quasispecies influencing HBeAg titer independent of viral load was also identified. These findings have practical implications for the use of quantitative serology as clinical biomarkers.

Acknowledgements

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

The authors thank Abbott Laboratories for provision of reagents and equipment; Professor Hans Tillmann for critically evaluating the data and providing useful comments; and Ms. Alison Boyd for assistance in performing the liver immunohistochemical studies.

References

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  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

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

Additional Supporting Information may be found in the online version of this article.

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HEP_23571_sm_Chinese-Version.pdf839KChinese Version

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