Research Article

Hepatitis B surface antigen: Relation to hepatitis Breplication parameters in HBeAg-negative chronic hepatitis B

Emanuel K. Manesis1,⇑, George V. Papatheodoridis2, Dina G. Tiniakos3, Emilia S. Hadziyannis2,Olga P. Agelopoulou3, Thalia Syminelaki3, Christos Papaioannou2, Theodoros Nastos4,

Peter Karayiannis4

1Division of Internal Medicine, Athens University Medical School, Athens, Greece; 2Academic Department of Medicine,Athens University School of Medicine, Hippokration General Hospital, Athens, Greece; 3Laboratory of Histology and Embryology,

Athens University Medical School, Greece; 4Department of Medicine, Hepatology Section, Division of Medicine, Imperial College, London, UK

Background & Aims: Translation of HBsAg depends on transcrip- Conclusions: In untreated HBeAg-negative CHB, serum HBsAg

tion of the appropriate mRNAs from cccDNA, but its relation toother hepatitis B virus (HBV) replication parameters is notknown, inasmuch as integrated sequences of HBV-DNA may alsocontribute to its serum levels, especially in HBeAg-negativechronic hepatitis B (CHB) patients.Methods: We investigated HBsAg serum levels, its hepatocellularexpression, and their relationship to HBV replicative- and host-response parameters before treatment in 54 HBeAg-negativeCHB patients and in 15 of them after 40.1 ± 33.3 months of viro-logical response on oral antiviral (NUC) therapy also. LivercccDNA and HBV-DNA quantitation, HBsAg- and HBcAg-immuno-staining were performed in the same needle biopsy material,while serum HBsAg and HBV-DNA levels were measured in sam-ples drawn on the day of liver biopsy.Results: In untreated patients, serum HBsAg correlated positivelywith HBsAg-positive hepatocytes/mm2 (p = 0.003) and weaklywith serum HBV-DNA, but not with cccDNA, liver HBV-DNA,HBcAg-positive hepatocytes/mm2, or ALT. cccDNA correlated sig-nificantly with liver HBV-DNA (p <0.00001), ALT (p = 0.001), andserum HBV-DNA levels (p = 0.012) but not with liver HBsAg orHBcAg. Antiviral therapy decreased serum HBsAg levels by79.6% (p = 0.012) and liver HBV-DNA by 84.4% (p = 0.026) inpaired comparisons and, as expected, significantly decreasedserum HBV-DNA and ALT levels, but not cccDNA.

Journal of Hepatology 20

Keywords: Serum HBsAg levels; HBeAg-negative chronic hepatitis B; cccDNA;Total liver HBV DNA; Serum HBV DNA; HBsAg-immunostaining; HBcAg-immunostaining.Received 1 June 2010; received in revised form 30 September 2010; accepted 6October 2010; available online 5 December 2010⇑ Corresponding author. Address: 19 Mavromateon Street, Athens 104 34,Greece. Tel.: +30 210 8829023; fax: +30 210 8210257.E-mail address: emanesis@med.uoa.gr (E.K. Manesis).Abbreviations: ALT, alanine aminotransferase; cccDNA, covalently closed circularDNA; CHBe�, HBeAg-negative chronic hepatitis B; CHBe+, HBeAg-positive chro-nic hepatitis B; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B ‘‘e’’ antigen;HBsAg, hepatitis B surface antigen; HBV DNA, hepatitis B viral DNA; HBV, hepa-titis B virus; HDV, hepatitis delta virus; HIV, human immunodeficiency virus;IFNa, alpha interferon; IQR, interquartile range; IU/L, international units per liter;15 IU/ml, international units per milliliter; mRNA, messenger RNA; NUC, nucle-os(t)ide analogue; D, difference.

levels reflect liver HBsAg, but not cccDNA or liver HBV-DNA, sug-gesting that they are not solely dependent on the replicative cycleof HBV. Effective NUC therapy for 3.34 years significantly lowersserum HBsAg and liver HBV-DNA, but not cccDNA.� 2010 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Introduction

Hepatitis B surface antigen (HBsAg) was the first hepatitis B virus(HBV) protein to be detected in serum [1] and since its discoveryit has been the hallmark of active HBV infection. Loss of serumHBsAg, spontaneously or following treatment, represents the bestpossible outcome in HBV-infected patients [2]. However, this isnot equivalent to complete HBV eradication since residual virusin the form of covalently closed circular DNA (cccDNA) may per-sist in a few hepatocytes for many years, even after HBsAg loss orseroconversion [3]. HBsAg levels have been traditionally mea-sured in serum qualitatively. However, the recent availability ofa quantitative test using newer-generation autoanalyzers andexpression of its levels in standardized international units perml [4] has provided renewed interest in the value of this time-honored marker. Indeed, recent evidence supports the role ofquantitative measurements of HBsAg as a tool to predict outcome[5,6] or to tailor the duration of alpha-interferon treatment [7]and possibly, in patients with long-term virological response tonucleos(t)ide (NUC) analogues, to act as a marker of duration oftreatment or even as a stopping rule for treatment [5]. Inuntreated patients, serum HBsAg levels are higher in HBeAg-posi-tive than in HBeAg-negative chronic hepatitis B (CHB) patients[8] and correlate with serum HBV-DNA levels [5,8]. In treatedpatients, a sharp decrease of serum HBsAg levels is observed inpatients responding to interferon-alpha (IFNa) with a muchslower drop in those responding to NUCs [5,9].

On the other hand, cccDNA, the dormant depot of HBV infec-tion, is also higher in HBeAg-positive CHB patients, associatedwith enhanced HBV replication, while it is lower in HBeAg-negative CHB patients and inactive carriers [10–13], indicatinga possible association with serum HBsAg levels. The intense

11 vol. 55 j 61–68

Research Article

interest for a possible correlation of these two parameters isstrengthened by the fact that cccDNA determination requires aliver biopsy and specialized molecular biology techniques avail-able only in a few laboratories, while HBsAg can be routinelymeasured in serum by common autoanalyzers. Indeed, previouswork has shown a correlation between cccDNA and serum HBsAgin CHB patients not discriminating between HBeAg-positive orHBeAg-negative cases [10,13]. A parallel reduction of bothparameters has also been demonstrated in patients receivingNUC therapy alone or in combination with pegylated IFN[9,10,14]. However, in the case of HBeAg-negative CHB, recentevidence indicates that there is no association between serumHBsAg and liver cccDNA [15,16].

The above findings underline the need for further research onthe origin of serum HBsAg. Is HBsAg directly related to HBV rep-lication? Is it really related to liver cccDNA content? Why doHBsAg serum levels persist during virological suppression byNUCs? Are there extrareplicative sources for HBsAg productionand what is their relative contribution? With these questions inmind we proceeded to investigate serum and liver tissue viraland host parameters related to HBV replication in a group ofuntreated HBeAg-negative CHB patients. In some of them, wealso studied the same parameters before and during long-termvirological response to NUCs.

Patients and methods

Patient population

We studied 54 randomly selected patients [35 males, 19 females; respectivemedian (IQR) age 48.9 (42.8–58.2) and 57.5 (33.1–66.6), p = NS] with HBeAg-negative CHB, followed at the Hepatology Outpatient Clinic, Academic Depart-ment of Medicine, Hippokration General Hospital, Athens, Greece. All patientswere native Greeks, almost all of them (96%) infected with HBV genotype Dand carrying a pre-core mutation [17,18]. All patients had a liver biopsy beforestarting treatment and fulfilled the following inclusion criteria: serological andhistological confirmation of HBeAg-negative CHB [19] and available liver tissuestored at �70 �C immediately after liver biopsy. No patient had evidence ofHCV, HDV, or HIV coinfection or hepatocellular carcinoma. All patients weretreatment-naive, except for 19 (35%) who had not responded or had relapsedafter a previous course of recombinant IFNa, which had been discontinued17.9 (7.8–50.9) months before the baseline liver biopsy. Fifteen of the 54patients [8 males, 7 females; median (IQR) age 51.7 (45.2–59.1) and 47.1(38.2–66.7), respectively (p = 0.955)] were being treated with NUC’s [lamivu-dine (LAM): 6, adefovir (ADF) 2, lamivudine plus adefovir (LAM + ADF): 2, tel-bivudine (LdT): 5] and had available an appropriately stored repeat liverbiopsy. During NUC treatment, all patients were followed-up with liver func-tion tests performed every three months and serum HBV DNA determinationsevery six months or more frequently, if needed. Only patients with serum HBVDNA undetectable by a sensitive quantitative polymerase chain reaction (PCR)technique throughout treatment and to repeat liver biopsy were includedin the study. Repeat liver biopsies were performed at 40.1 ± 33.3 monthsafter starting treatment. The duration of treatment varied with the type ofNUC. For LAM, LdT, and ADF-based treatment the median (IQR) treatmentduration was 36.0 (9.8–67.7), 13.0 (12.8–13.1), and 64.3 (19.9–92.1) months,respectively.

The study was approved by the Ethical Committee of the Hospital.

Liver histology and immunohistochemistry

Baseline and on-treatment liver biopsies were evaluated in batches, blindly by anexperienced liver histopathologist (DGT) and scored for necroinflammationand stage of fibrosis according to Ishak et al. [20]. The median (IQR) length of the54 baseline liver biopsies was 16.5 (13.6–25.0) mm and that of repeat liver biopsiesin the 15 NUC-treated patients, 18 (16.0–32.0) mm. Non-cirrhotic liver biopsies con-tained a mean number of 13.7 (range 10–21) portal tracts. The number of hepatocytes

62 Journal of Hepatology 20

per mm2 of liver biopsy depended on the amount of non-parenchymal tissue presentin that area (i.e. portal tracts, fibrous septa). In our material, it was estimated to be�700 hepatocytes/mm2.

Immunohistochemical staining for HBsAg was performed with a commercialanti-HBs mouse monoclonal antibody and an anti-HBc rabbit polyclonal antise-rum (T9 and Ab1, respectively, Thermo Scientific, MA, USA) using the Novolink™polymer detection system (Novocastra Laboratories Ltd, UK). The primary anti-bodies were applied at a 1:25 (anti-HBs) and 1:50 (anti-HBc) dilution overnightat 4 �C and the final reaction was developed using 30 ,30-diaminobenzidin (DAB)(Sigma Aldrich, St. Louis, MO, USA) as chromogen. The total length and widthof the tissue cores from each liver biopsy were measured and the tissue areawas calculated in mm2. The total number of HBsAg- and HBcAg-positive hepato-cytes was counted in each biopsy by two observers (OPA and TS) using a double-headed microscope at 40� magnification. The number of HBsAg- and HBcAg-positive hepatocytes/mm2 was calculated in each case and results were subse-quently verified by the liver histopathologist (DGT). Additionally, the subcellularlocalization of HBsAg (membranous, cytoplasmic, nuclear) and HBcAg (nuclear,cytoplasmic, or both), the intensity (mild, moderate, strong), the topography (aci-nar zone involvement), and the distribution pattern (isolated, clusters, diffuse) ofpositive immunostaining were recorded in each case.

Serum HBsAg and HBV DNA measurements

Blood was collected on the day of liver biopsy and sera were prepared and storedimmediately at �20 �C and thawed once. Serum HBsAg levels were quantifiedusing the Abbott Architect HBsAg QT assay [4]. The Architect HBsAg assay usesmicroparticle-coated monoclonal anti-HBs and after adding an acridinium-labeled anti-HBs conjugate, the resulting chemiluminescence is measured in rel-ative light units. A direct relationship exists between the amount of HBsAg in thesample and the relative light units detected by the Architect optical system. Theconcentration of HBsAg in IU/ml in the specimen is determined using an ArchitectHBsAg calibration curve with a range of 0.05–250 IU/ml. Samples are finallydiluted at 1:20 and 1:320 in order to expand the upper limit of the dynamic rangefrom 250 to 80,000 IU/ml.

Serum HBV-DNA levels were also measured in the same serum specimens asHBsAg. Measurements were done using the Cobas TaqMan HBV Test (Roche Diag-nostics, Indianapolis, IN, USA), with a lower quantitation limit of 100 copies/ml(20 IU/ml). HBV DNA results were expressed in copies/ml or in their respectivelog10 derivative.

Liver cccDNA and total HBV DNA measurements

Portions of liver biopsies taken for light microscopy were stored at �70 �C imme-diately after the procedure. These were used to extract total cellular HBV DNAthat included cccDNA and relaxed circular double stranded DNA (RC-ds DNA),as well as genomic DNA. For this purpose, the MasterPure™ complete DNA andRNA purification kit (Epicentre Biotechnologies, Cambio Ltd., Cambridge, UK)was employed according to the tissue sample extraction protocol, as detailedby the manufacturer. The extracted DNA was subjected to real time PCR amplifi-cation using specific primers for the detection of total HBV-DNA, cccDNA, and b-globin. The primers and cycling conditions for total HBV DNA detection were asreported previously [21]. Primers cccDNA(+) (50-CCGTCTGTGCCTTCTCATCTGC-3,positions 1547–1568) and cccDNA(�) (50-CAAAGCCACCCAAGGCACAG-3, posi-tions 1877–1896) which amplified only cccDNA were used for its detection underthe same conditions as for total HBV DNA [21]. A standard curve, obtained from aserially diluted HBV containing plasmid, was used to quantitate cccDNA and totalHBV-DNA. Beta-globin DNA was detected by using the LightCycler� control DNAkit from Roche Diagnostics (Lewes, East Sussex, UK), and the quantitative resultsobtained were used to estimate the cell number in the biopsies used as reportedby others [13]. The results of cccDNA and total HBV-DNA were then expressed incopies per cell.

Statistical analysis

Data entry and statistical comparisons were made with the statistical packageSPSS (version 15.0, SPSS Inc., Chicago, IL, USA). For bivariate correlations, theSpearman’s rho test was used. Categorical data were compared by the chi squaretest. The t-test was used for quantitative parametric data comparison and theMann–Whitney and the Kruskal–Wallis tests for non-parametric data. For pairedcomparisons the paired t-test or the Wilcoxon’s signed ranks test were used, asappropriate. All laboratory measurements were reported as median (IQR), unlessotherwise stated. A two-tailed p value of <0.050 was considered to be statisticallysignificant.

11 vol. 55 j 61–68

JOURNAL OF HEPATOLOGY

Results

Interrelations of baseline virological and host-related measurements

The main patient characteristics and values for all parameterstested are shown in Table 1. cccDNA and total liver HBV DNAcould be detected in all liver biopsies of untreated patients, witha median (IQR) content of 0.3 (0.1–1.1) and 2.8 (0.9–10.5) copies/cell, respectively. In general, 30.6% and 69.4% of the patients car-ried P1 copy/cell of cccDNA and total HBV DNA, respectively,while 24.5% of the patients harbored P10 copies/cell of totalHBV DNA in their liver. Considering that hepatocytes constitute70–80% of cells found in the liver [22], the median cccDNA copynumber would be approximately 1 copy per 2 hepatocytes (or17 copies per one HBcAg-positive hepatocyte) and the totalHBV DNA, 4–5 copies per hepatocyte (or approximately 154 cop-ies per HBcAg-positive hepatocyte).

Patients at the upper quartile of age (>59.5 years old) com-pared to younger patients (lowest quartile, <42.4 years of age)had significantly higher stage of liver fibrosis (p = 0.014). Other-wise, sex and age had no effect on serum HBsAg or on any otherparameter tested. Patients at the upper quartile of necroinflam-mation grade (P9) had, as expected, higher serum HBV DNAand ALT (p <0.00001) and also, higher total liver HBV DNA (14.5vs. 0.9 copies/cell, p = 0.004) and more cccDNA (0.5 vs. 0.1 cop-ies/cell, p = 0.034) content, compared to those of the lowestquartile (66). Presence of cirrhosis was associated with a highergrade of necroinflammation (p = 0.008), but with less cccDNAcontent of the liver (median 0.2 vs. 0.4 copies/cell, p = 0.046).The necroinflammatory score, the stage of liver disease and thepresence or absence of cirrhosis had no significant effect on thebaseline levels of serum HBsAg.

Table 1. Characteristics of patients at baseline and with oral antiviral therapy.⁄

Age (years) a

Sex [males (%)]Histology c

Grade a,c

Stage a,c

Cirrhosis [n (%)] d

Serum ALT (IU/L) a

HBsAg (Log10 IU/ml) a

HBV DNA (copies/ml) a

Liver HBV DNA (copies/cell) a

cccDNA (copies/cell) a

HBsAg (positive cells/mm2)a

HBcAg (positive cells/mm2)a

Treatment duration (months)

8 (6-9)3 (2-4)11 (20.4%)

91 (39-181)3.157 (2.501-3.519)6.396 (4.611-7.610)

2.8 (0.9-10.5)0.3 (0.1-1.1)224 (10-373)2.9 (0-62.7)

49.7 (42.4-59.5) 4

Variables All PatientsBaseline(N = 54)

B(

35 (64.8%)

NA e

⁄All patients were native Greeks, 96% of them infected with of HBV genotype D carryinaMedian (interquartile range).bPaired t-test.cAccording to Ishak et al. [20].dIshak’s stage 5 or 6.eNot applicable.

Journal of Hepatology 20

Patients with baseline HBV-DNA levels of <5000 copies/ml(N = 10) had significantly lower serum HBsAg levels (2.393 vs.3.261 log10 IU/ml, p = 0.024), immunohistochemical expressionof HBsAg (3.3 vs. 234.4 positive cells/mm2, p = 0.012), liver HBVDNA (0.08 vs. 3.15 copies/cell, p = 0.016) and borderline lowcccDNA content of the liver (0.1 vs. 3.5 copies/cell, p = 0.065)compared to patients with P5000 copies/ml (N = 44).

Correlation of serum HBsAg and HBV and host parameters

Serum HBsAg levels presented a weak but significant, positivecorrelation with serum HBV DNA (r = 0.298, p = 0.029) (Table 2,Fig. 1). Liver HBV DNA correlated significantly with cccDNA (p <0.00001), ALT (p <0.0001), serum HBV-DNA levels (p = 0.0003),and the number of HBcAg-positive cells (p = 0.042), while cccDNAcorrelated only with total liver HBV DNA, serum HBV DNA(p = 0.012), and ALT (p = 0.001) (Table 2). Comparison of the high-est and lowest (upper and lower quartile) cellular contents ofcccDNA in baseline liver biopsies was not associated with signifi-cant differences in serum and liver HBsAg (p = 0.124 and p = 0.594,respectively), but total liver HBV DNA and serum ALT were signif-icantly higher in patients harboring higher cellular cccDNA con-tent (23.8 vs. 0.2 copies/cell, p = 0.001; 110 vs. 30 IU/L, p = 0.002,respectively) (Fig. 2). Also, the liver HBV DNA production rate,expressed as the product of liver HBV DNA per unit of cccDNAper cell, did not correlate significantly with either the serum levelsor the liver expression of HBsAg (p = 0.097 and 0.917, respec-tively), but it did correlate with HBcAg (p = 0.031).

The necroinflammatory score correlated significantly with thenumber of HBcAg-positive hepatocytes (r = 0.389, p = 0.015), ALT(r = 0.430, p = 0.001), serum HBV DNA (r = 0.290, p = 0.034), andtotal liver HBV DNA (r = 0.305, p = 0.033), but not with serum

8 (8-10)3 (2-4)1 (6.7%)

180 (114-252)3.363 (2.713-3.761)7.035 (6.305-8.018)

8.95 (2.65-83.00)0.70 (0.18-1.93)224 (31-401)25.6 (0.75-66.4)

8.6 (44.2-59.9) 55.6 (50.8-62.3)

4 (3-4)2 (1-3)1 (6.7%)

24 (17-27)2.672 (2.207-3.134)<2.000 (<2.000-2.697)

1.40 (0.90-5.90)0.40 (0.06-1.20)93 (19-192)29.1 (0.001-109.0)

0.0010.0121.000

<0.00010.012<0.0001

0.0260.4270.9420.255

0.436

Nucleos(t)ide-Analogue-Treated Groupefore

N = 15)End(N =15)

p Value(Paired)b

8 (53.3%)

40.1 ± 33.3

g the pre-core mutation.

11 vol. 55 j 61–68 63

Seru

m H

BV-D

NA

(cop

ies/

ml)

10,000,000,000

1,000,000,000

100,000,000

10,000,000

1,000,000

100,000

10,000

1000

100

10

1

0

Tota

lliv

er H

BV D

NA

(cop

ies/

cell)

100

10

1

0

10,00010001000 1 01

HBs

Ag-p

ositi

ve c

ells

/mm

2

10,000

1000

100

10

10

Serum HBsAg levels (IU/ml)10,00010001000 1 01

Serum HBsAg levels (IU/ml)

10,00010001000 1 01Serum HBsAg levels (IU/ml)

10,00010001000 1 01Serum HBsAg levels (IU/ml)

R2 = 0.237p = 0.003

R2 = 0.002p = 0.265

R2 = 0.151p = 0.029

R2 = 0.005p = 0.010

Live

r ccc

DN

A (c

opie

s/ce

ll)

A B

C D

Fig. 1. Correlation between HBsAg serum levels and parameters of HBV replication. A significant and close correlation is noted between serum HBsAg levels and thenumber of HBsAg-positive hepatocytes/mm2 (A) and serum HBV-DNA levels (B), but not with liver cccDNA (C) and total liver HBV DNA (D). Straight lines denote theregression of the x-axis over the y-axis values and curved lines, the 95% confidence intervals.

Table 2. Correlation between baseline virological and host parameters in patients with HBeAg-negative chronic hepatitis B. a

Serum HBsAg 1

(Log10 )Liver HBsAg 2

(Log10)Liver HBcAg 2

(Log10)Serum HBV DNA 3

(Log10)Serum ALT 4

(Log10)Liver cccDNA 5

(Log10)

p = 0.003, r = 0.465 p = 0.154, r = 0.233

p = 0.341, r = 0.157

p = 0.029, r =0.298

p = 0.025, r = 0.364

p = 0.052, r = 0.313

p = 0.323, r = 0.137

p = 0.424, r = 0.130

p = 0.059, r = 0.305

p <0.0001, r = 0.607

p = 0.265, r = -0.161

p = 0.609, r = -0.087

p = 0.360, r = 0.157

p = 0.012, r = 0.356

p = 0.001, r = 0.468

p = 0.948, r =-0.010

p = 0.754, r = 0.053

p = 0.042, r = 0.341

p = 0.0003, r = 0.501

p <0.0001, r = 0.590

p <0.00001, r = 0.736

Parameter Liver HBsAg(Log10)

Liver HBcAg(Log10)

Serum HBV DNA(Log10)

Serum ALT(Log10)

Liver HBV DNA (Log10)

Liver cccDNA(Log10)

aPearson’s correlation.1IU/ml. 2Number of positive hepatocytes per mm2 of liver tissue. 3Copies per ml. 4IU/L. 5Copies per cell.

Research Article

64 Journal of Hepatology 2011 vol. 55 j 61–68

4th3rd2nd1st

Tota

l liv

er H

BV D

NA

(cop

ies/

cell)

100

10

10

4th3rd2nd1st

Seru

m A

LT (I

U/L

)

1000

100

10

p = 0.001 p = 0.002

Liver cccDNA (copies/ml) QuartilesLiver cccDNA (copies/ml) QuartilesLiver cccDNA (copies/ml) Quartiles

A B C

4th3rd2nd1st

Seru

m H

BsAg

leve

ls (I

U/L

) 10,000

1000

100

10

10

p = 0.124

Fig. 2. Changes in total liver HBV DNA and serum HBsAg and ALT levels across increasing liver cccDNA quartiles. A significant increase is observed in total liver HBVDNA content (A) and serum ALT (B), but not in serum HBsAg levels (C).

JOURNAL OF HEPATOLOGY

HBsAg or other parameters tested. The histological stage and thepresence of cirrhosis, besides a significant correlation with thenecroinflammatory score (r = 0.508, p <0.00001), were not relatedto other parameters.

Liver immunohistochemistry

The median (IQR) count of HBsAg- and HBcAg-positive hepato-cytes (Fig. 3) was 224 (10–373) and 2.9 (0–63) per mm2 of livertissue, respectively, or 32.0% (1.4–52.3%) and 0.4% (0–9.0%) ofthe total number of hepatocytes per liver biopsy, respectively. Asignificant positive correlation was observed between HBsAg-positive hepatocytes/mm2 and HBsAg serum levels (r = 0.465,p = 0.003). Moreover, the extent of HBsAg-positive immunostain-ing (negative staining; isolated positive cells; positive cells inclusters; diffusely positive cells) was associated with increasingmedian HBsAg serum levels (2.364, 2.622, 3.135, 3.553 log10 IU/ml, or 231, 419, 1366, 3576 IU/ml, respectively; p = 0.001) and asimilar significant relationship was observed between the inten-sity of HBsAg immunostaining (negative, mild, moderate, strong)and the respective median HBsAg serum levels (2.364, 3.115,3.262, 3.368 log10 IU/ml, or 231, 1303, 1826, 2336 IU/ml;p = 0.012). As also observed with serum HBsAg, a weak associa-tion was present between liver HBsAg and serum HBV DNA, butthere was no association of the former with ALT, cccDNA, liverHBV DNA or the number of HBcAg-positive hepatocytes permm2 of liver tissue (Table 2).

Fig. 3. Liver Immunohistochemistry for HBsAg and HBcAg. (A) Diffuse HBsAg-specific cytoplasmic (100�) and (B) diffuse HBcAg-specific nuclear immuno-staining (200�) in liver biopsies of patients with HBeAg-negative chronichepatitis B.

Journal of Hepatology 20

In liver biopsies with immunohistochemically identifiableHBsAg, pure membranous HBsAg immunostaining was notobserved in any case, while cytoplasmic or mixed cytoplasmic-plus-membranous localization was present in 38.5% and 41.0%of the 54 baseline biopsies, respectively. The type of HBsAg local-ization per se, had no statistical impact on any of the serum orliver parameters tested. However, presence of either cytoplasmicor cytoplasmic-plus-membranous HBsAg localization was associ-ated with significantly higher baseline serum HBsAg levels whencompared to cases lacking HBsAg immunostaining (p = 0.003).Serum HBV DNA was also significantly higher in cases with cyto-plasmic-plus-membranous localization vs. no HBsAg immuno-staining (p = 0.009).

The number of HBcAg-positive hepatocytes per mm2 corre-lated weakly with liver HBV DNA (p = 0.042) and less so withserum HBV DNA and ALT (p = 0.052 and 0.059, respectively),but not with liver cccDNA (Table 2). The presence of nuclearand/or cytoplasmic HBcAg localization was significantly associ-ated with higher median serum HBV DNA (6.519 vs. 5.190log10 copies/ml; p = 0.035) and ALT levels (85 vs. 59 IU/L;p = 0.034), but not with serum or liver HBsAg, cccDNA, or liverHBV DNA. The acinar localization of HBcAg-positive hepatocytesand the intensity of HBcAg immunostaining were not associatedwith significant differences of serum HBsAg, HBV DNA or ALT lev-els, or of liver HBV DNA and cccDNA, or the number of HBsAg-positive hepatocytes/mm2.

The effect of nucleos(t)ide analogue treatment

In the group with 15 NUC-treated cases (8 males, 7 females), thefemale patients appeared to have more active disease with signif-icantly higher median cccDNA (7.0 vs. 1.7 copies/cell, p = 0.027)and total liver HBV DNA (79.0 vs. 2.90 copies/cell, p = 0.004) com-pared the 8 male patients, although their respective median his-tological grade (10 vs. 8), ALT (195 vs. 147) and serum HBV DNA(7.875 vs. 6.808 log10 copies/ml) did not reach statistical signifi-cance. Other baseline and on-treatment characteristics of treatedpatients are shown in Table 1 and the temporal changes of serumHBsAg, HBV DNA, and ALT in Fig. 4A. On NUC treatment, the med-ian baseline ALT was normalized and HBV DNA became undetect-able in all patients. In paired comparisons, the intrahepatic HBVDNA productivity (expressed as the log10 of total liver HBVDNA per cccDNA copy) and serum HBV DNA production rate

11 vol. 55 j 61–68 65

A B

10

100

1000

10,000

100,000

1,000,000

10,000,000

100,000,000

0 6 12 18 24 30 36 42 48 54 60 66Time (months of treatment)

Nucleos(t)ide Analogue Treatment

Live

r ccc

DN

A (c

opie

s pe

r cel

l)

10

1

0Tota

l liv

er H

BV D

NA

(cop

ies

per c

ell) 1000

100

10

10

Seru

m H

BsAg

(IU

/L)

100,000

10,000

1000

100

10

Before

Treatm

ent

On Trea

tmen

t

Before

Treatm

ent

On Trea

tmen

t

Before

Treatm

ent

On Trea

tmen

t

a b c

p = 0.012 p = 0.427p = 0.026

HBV DNA

ALTHBsAg

Seru

m H

BsAg

(IU

/ml),

HBV

DN

A (c

/ml),

AL

T (IU

/L)

Fig. 4. Nucleos(t)ide analogue treatment results. (A) Temporal changes of serum HBsAg, HBV DNA, and ALT levels of 15 patients during nucleos(t)ide analogue treatment.Besides the non-detectablity of HBV DNA and ALT normalization, a gradual decrease in serum HBsAg levels is noted, which attains statistical significance in pairedmeasurement comparisons in individual patients. (B) Baseline and last on-treatment paired comparisons of serum HBsAg, total liver HBV DNA, and cccDNA in the samepatients on nucleos(t)ide analogue treatment. Serum HBsAg (a) and total liver HBV DNA content (b) were significantly reduced, while liver cccDNA (c) remained statisticallyunchanged. (p values of log10-transformed data).

Research Article

(expressed as the log10 of serum HBV DNA per cccDNA copy)decreased significantly before and on NUC treatment (p = 0.034and p <0.00001, respectively). In paired measurements, also, themean log10 baseline serum HBsAg levels decreased significantly,from 3.363 to 2.672 (or 2305 to 470 IU/ml, respectively;D = �79.6%; p = 0.012) and a similar significant decrease wasnoted in total liver HBV DNA (from 8.95 to 1.40 copies/cell,respectively; D = �84.4%, p = 0.026), but not in liver cccDNA(p = 0.427) and in the number of HBsAg- or HBcAg-positive hepa-tocytes per mm2 of liver tissue (p = 0.942 and p = 0.255, respec-tively) (Fig. 4B). In this cohort, the mean decrease of serumHBsAg on NUCs was 550 IU/ml per year.

Except for the significant suppression in serum HBV DNA andALT observed with all individual NUCs used in this study, LAM,LdT, and ADF-based treatment induced significant improvementin the histological grade (p = 0.006, p = 0.009, p <0.0001, respec-tively), but had no effect on the stage of liver disease. Paired com-parisons of serum HBsAg levels and the number of HBsAg- andHBcAg-positive hepatocytes were significantly suppressed onlyby LAM (p = 0.020, p = 0.015, p = 0.031, respectively). ADF-basedtreatment had a borderline suppressive effect on liver cccDNA(p = 0.055) and LdT had a borderline suppressive effect on totalliver HBV DNA (p = 0.065]. However, the number of patients trea-ted with individual NUCs was too small and the duration of treat-ment varied for valid conclusions.

Discussion

Findings in this study indicate that serum HBsAg levels inpatients with HBeAg-negative CHB are not associated with mark-ers of HBV replication, i.e. cccDNA, total liver HBV DNA content orthe cellular HBV DNA production rate, but they are closely asso-ciated with the number, the immunostaining intensity, and thedistribution pattern of HBsAg-positive hepatocytes per mm2 ofliver tissue. Also, in cases with increased histological activity, adiscrepancy has been found between enhanced HBV replication

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without significant changes in serum HBsAg levels. Finally, NUCtreatment with non-detectable long-term serum HBV DNA wasnot associated with parallel elimination of serum HBsAg. Takentogether, the above indicate that in HBeAg-negative patients, adissociation is present between HBsAg production and HBV rep-lication. These findings are in agreement with recently publisheddata demonstrating a close correlation between serum HBsAgand HBV replication in HBeAg-positive patients, but not inHBeAg-negative CHB patients [16,23].

The core element in the life cycle of HBV replication iscccDNA. It persists in the nuclei of infected hepatocytes form-ing stable minichromosomes and acts as a template for thegeneration of the pregenomic mRNA, pre-core mRNA, and allother subviral mRNAs, including those for the surface antigenproteins [24,25]. It is well established that the cccDNA contentof the liver varies in the different phases of the natural courseof chronic HBV infection and that cccDNA levels are signifi-cantly higher in HBeAg-positive, compared to HBeAg-negativepatients with CHB [10–13]. A low level of liver cccDNA contentwas also found in this study in accordance with other studies[10,13].

Higher levels of cccDNA in the liver tissue are not necessarilyassociated with increased HBV transcription. Transcription ofcccDNA can be suppressed or enhanced by several intrinsic fac-tors, such the degree of cccDNA methylation or the acetylationof the surrounding histones, respectively [26–31], as well as epi-genetic factors such as intranuclear accumulation of core and Xproteins which may bind cccDNA molecules [32]. The negativeeffect of methylation may affect HBV transcription as a whole[28,29] or suppress the expression of some of the subviral mRNAs[30]. Methylation may have the same effect not only on cccDNA-related transcription, but also on transcription of HBV fragmentsintegrated into the host genome and coding for viral proteins,including HBsAg [33]. Currently, it is not known whether cccDNAmethylation is enhanced during the HBeAg-negative phase, andwhether it differentially interferes with the whole HBV genomevs. HBsAg transcription.

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Early in the natural course of HBV infection, serum HBsAg lev-

els are high [8,34], closely associated and reflecting the enhancedHBV replication rate of the HBeAg-positive phase [16]. However,later in the HBeAg-negative phase, HBsAg levels become appre-ciably lower [35] and, as our data and those of other studies haveshown [16,23], HBsAg serum levels are dissociated from HBV rep-lication. Our study and those of others [16,23] do not provide anydirect, indisputable evidence for the nature of this alternativesource of HBsAg production, if any. It is possible that no alterna-tive source exists and, the apparent discrepancy is only due toredirected subviral mRNA production, as a viral adaptation toincreased immunological pressure during the HBeAg-negativephase [36]. An alternative explanation is that HBsAg transcriptionfrom fragments of the HBV genome integrated into the host chro-mosomes [37,38]. Indeed, random HBV viral integration into thehost genome has been observed to begin even at very early stagesin HBV infection [39,40] and sequences of the S genes are oftenpresent in the integrated segments [41]. HBeAg-negative CHBpatients usually have a longer, mostly perinatal HBV infectionhistory [36] and are expected to have more extensive HBV DNAintegration than HBeAg-positive ones, who usually have a shorterduration of infection. However, direct proof of this hypothesis islacking, since to date no method exists for reliable differentiationbetween surface gene mRNAs of integrated vs. those of cccDNAorigin.

NUC treatment does not directly affect the cccDNA content ofthe liver. Decreases in cccDNA levels are derived from the lack ofsufficient re-entry of viral nucleocapsids to the nucleus, due tostrong inhibition of viral DNA synthesis in the cytoplasm, andfewer incoming viruses from the blood. Following this scenario,NUC administration will lead to cccDNA depletion after manyyears of treatment. Data from this study present evidence thatlong-term virological response induced by LAM, LdT, and ADF-based treatment, besides the expected non-detectable serumHBV DNA and even normalization of ALT, leads to a significantdecrease in the intrahepatic HBV DNA production rate, the totalHBV DNA content of the liver and also to serum HBsAg levels.This work did not provide evidence for the duration of NUC treat-ment in HBeAg-negative CHB patients, but suggested that suchtreatment is on good course and should continue, although3.3 years of virological response were probably not sufficientfor sustained off-treatment complete response. Serial measure-ments of serum HBsAg levels may be helpful in this respect.

Our data also provide limited insight on the possible differen-tial effect of various NUCs. Three years of effective treatment withLAM had a significant suppressive effect on serum HBsAg levels,the immunohistochemical expression of HBsAg and HBcAg inthe liver, but had no effect on the total liver HBV DNA or thecccDNA content. Treatment of a shorter duration (median13 months) with the more potent HBV polymerase inhibitorLdT had no effect on cccDNA but was associated with borderlinesuppression of total liver HBV DNA. Longer treatment with anADF-based regime (median 5.36 years) was associated with bor-derline cccDNA suppression. Although some of the above findingsare in line with previous observations [10], our data cannot leadto solid conclusions as to the differential effect of various NUCs,due to the limited number of cases studied.

Data presented in this article provide a useful piece of infor-mation for the life-cycle of HBV and the effects of treatment inHBeAg-negative CHB patients, who today represent the largestgroup of HBV-infected people worldwide [42]. We have shown

Journal of Hepatology 20

that, although apparently dissociated from the replicative cycleof HBV, HBsAg quantitatively measured in the serum decreasessignificantly after several years of effective NUC therapy. In anew role, serum HBsAg could provide the only easily measurableHBV product in treated HBeAg-negative patients, used as a stop-ping rule of NUC therapy.

Conflict of interest

The authors who have taken part in this study do not have a rela-tionship with the manufacturers of the drugs involved either inthe past or present and did not receive funding from the manu-facturers to carry out their research.

Financial support

This work was partially supported by a grant from Gilead Sci-ences Inc., and Roche (Hellas) S.A.

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