Distinct evolution and predictive value of hepatitis B virus precore and basal core promoter mutations in interferon-induced hepatitis B e antigen seroconversion

Distinct Evolution and Predictive Value of Hepatitis BVirus Precore and Basal Core Promoter Mutations in

Interferon-Induced Hepatitis B e AntigenSeroconversion

Hung-Chih Yang,1,2,3 Chi-Ling Chen,2 Yueh-Chi Shen,1 Cheng-Yuan Peng,4 Chun-Jen Liu,2,3 Tai-Chung Tseng,5

Tung-Hung Su,2,3 Wan-Long Chuang,6 Ming-Lung Yu,6 Chia-Yen Dai,6 Chen-Hua Liu,2,3 Pei-Jer Chen,2,3,7

Ding-Shinn Chen,2,3,7 and Jia-Horng Kao2,3,5,8

Precore (PC) (G1896A) and basal core promoter (BCP) (A1762T/G1764A) mutations of thehepatitis B virus (HBV) genome often emerge in chronic hepatitis B (CHB) patients. Theirroles in hepatitis B e antigen (HBeAg) seroconversion induced by interferon (IFN) therapyremain controversial, partly because quantitative analysis for these mutants is lacking. Thisstudy aimed to develop a new assay to accurately quantify the PC and BCP mutant percen-tages and correlate their dynamic changes with IFN-induced HBeAg seroconversion inHBeAg-positive CHB patients. The PC and BCP mutant percentages were analyzed by poly-merase chain reaction (PCR)-pyrosequencing. Our results showed that this quantitativeassay for PC and BCP mutants achieved high accuracy (R2 > 0.99) within a range between10% and 90% mutants. We examined dynamic changes of the PC and BCP mutant percen-tages following IFN treatment in 203 HBeAg-positive CHB patients. By multiple logisticregression analysis, we found that the chance of HBeAg seroconversion increased by 2.2%(odds ratio [OR] 5 1.022, 95% confidence interval [CI]: 1.009-1.034, P 5 0.001) and2.3% (OR 5 1.023, 95% CI: 1.010-1.037, P 5 0.001) per 1% increase of the pretreatmentPC and BCP mutant percentages, respectively, after adjustment for other predictors. How-ever, only the pretreatment PC mutation percentage was significantly associated with HBeAgseroconversion with HBV DNA < 2,000 IU/mL (OR 5 1.030, 95% CI: 1.014-1.047, P <0.001). Furthermore, the mutant percentage of PC, but not BCP, in patients achievingHBeAg seroclearance with HBV DNA < 20,000 IU/mL increased significantly during IFNtreatment (P 5 0.039). Interestingly, patients with HBeAg seroconversion who had a highPC mutant percentage at the end of IFN treatment tended to exhibit high viremia after sero-conversion. Conclusion: Quantitative analysis of PC and BCP mutants can predict IFN-in-duced HBeAg seroconversion and demonstrate their distinct evolution patterns duringHBeAg seroconversion. (HEPATOLOGY 2013;57:934-943)

Chronic hepatitis B virus (HBV) infection canlead to a broad spectrum of clinical outcomes,ranging from inactive carriers to severe hepatic

complications, such as liver cirrhosis and hepatocellular

carcinoma.1 The natural course of individuals withchronic hepatitis B (CHB), particularly those acquiringHBV infection in the perinatal period or early child-hood, consists of three distinctive phases of disease:

Abbreviations: BCP, basal core promoter; CHB, chronic hepatitis B; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; IFN, interferon; PC, precore.From the 1Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan; 2Graduate Institute of Clinical Medicine, National

Taiwan University College of Medicine, Taipei, Taiwan; 3Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan; 4Department ofInternal Medicine, China Medical University Hospital, Taichung, Taiwan; 5Department of Internal Medicine, Buddhist Tzu Chi General Hospital Taipei Branch,Taipei, Taiwan; 6Faculty of Internal Medicine, College of Medicine Kaohsiung Medical University, Kaohsiung, Taiwan; 7Department of Medical Research, NationalTaiwan University Hospital, Taipei, Taiwan; 8Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan.Received May 6, 2012; accepted October 14, 2012.Supported by grants from National Taiwan University Hospital (101-S1878), National Science Council (NSC 100-2314-B-002-049-MY3), Liver Disease

Prevention & Treatment Research Foundation, National Research Program for Biopharmaceuticals (NRPB), and the Taiwan-LiverNet Consortium (NSC100-2325-B-002-052).

934

immune tolerant, immune clearance, and inactiveresidual phases.2 In some patients with inactive disease,reactivation of hepatitis B can occur.Hepatitis B e antigen (HBeAg) is an important

serological marker of the disease status of CHB. Theseroconversion of HBeAg, defined by the loss ofHBeAg and appearance of anti-HBe antibody, signifiesthe transition from the immune clearance to the inac-tive residual phase and is often accompanied by thereduction of HBV replication and remission of hepato-cyte injury.3 CHB patients who undergo HBeAg sero-conversion usually exhibit a favorable prognosis.4

Although HBeAg seroconversion has long been recog-nized as a critical event in the natural history of CHB,the underlying mechanisms remain largely elusive.Several virological factors have been demonstrated

to impact the progression of CHB, including viralgenotypes, serum HBV DNA levels, and certain natu-rally occurring mutants.5 Because of the error-pronenature of the HBV reverse transcriptase, HBV mutantsoften emerge during the long-term HBV infection pe-riod.6,7 Among these mutants, the precore stop codon(PC) and basal core promoter (BCP) mutants are bestcharacterized. The PC mutant exhibits a G-to-A muta-tion in the precore region of HBV genome of nucleo-tide 1896 (codon 28; TGG to TAG), which abolishesthe synthesis of HBeAg.8 The double mutations ofBCP often occur at nucleotide 1762 and 1764 with ‘‘Ato T’’ and ‘‘G to A’’ substitution, respectively.Both PC and BCP mutations have been reported to

be associated with HBeAg seroconversion.9,10 Thesemutants are more prevalent in HBeAg-negative patientsthan in those with HBeAg positivity. Of note, most ofthe prior studies analyzed these mutants using qualita-tive assays, like direct sequencing. Few studiesattempted to quantify the percentages of PC and/orBCP mutants in a population of HBV within a singleindividual.13–16 Besides, longitudinal analysis of thedynamic change of PC and BCP mutants is scarce.Therefore, the exact roles of these mutants duringHBeAg seroconversion remain unclear. Recently,Nie et al.12 quantitatively and extensively analyzed thedynamic evolution of PC and BCP mutants in the lon-gitudinal serum samples from 18 CHB patients. They

found a temporal correlation between the increase ofPC and/or BCP mutant frequency and HBeAg serocon-version, although the conclusion is limited by the smallsample size. Interestingly, a previous study also revealedthat PC mutation decreases the risk of HCC, whereasBCP mutation is associated with the development ofHCC,14 indicating the distinctive roles of these mutantsin the long-term outcome of CHB patients.Interferon (IFN) has both an antiviral and immuno-

modulatory effect. IFN-based therapy with conven-tional or pegylated IFN has been shown to enhanceHBeAg seroconversion in CHB patients.15 Around30%-40% of the HBeAg-positive CHB patients receiv-ing a finite course of IFN therapy achieve HBeAgseroconversion, whereas HBeAg seroconversion occursin less than 15% of untreated patients.3,15 Severalprior studies using qualitative assays investigated theroles of PC and BCP mutants in IFN-induced HBeAgseroconversion,16–20 but did not achieve consistentresults. Therefore, the exact role of these two muta-tions in HBeAg seroconversion remains controversial.Pyrosequencing is a new sequencing technology based

on the principle of sequencing-on-synthesis. It is a cheap,high throughput, and convenient method for analysis ofthe frequency of a particular nucleotide position among aheterogeneous population, and is suitable to quantify theviral mutants in a large number of samples. It has beenshown to have the ability to accurately determine the fre-quency of influenza and HBV mutants in a mixture ofwildtype and mutant viruses.21,22 In this study, we devel-oped a novel quantification assay using a pyrosequencingtechnique to measure the percentages of PC and BCPmutants in patients receiving IFN therapy and correlatethem with the likelihood of HBeAg seroconversion.

Patients and MethodsPatients. A total of 203 HBeAg-positive CHB

patients who had a pretreatment serum alanine amino-transferase (ALT) level over two times the upper limitof normal (ULN) and received a finite course of IFNtherapy were retrospectively enrolled. Among them, 42patients participated in a controlled trial comparingthe efficacy of IFN-a-2b with and without ribavirinbetween 1998 and 1999 at the National Taiwan

Address reprint requests to: Prof. Jia-Horng Kao, Distinguished Professor and Director, Graduate Institute of Clinical Medicine, National Taiwan UniversityCollege of Medicine, 7 Chung-Shan South Road, Taipei 10002, Taiwan. E-mail: [email protected]; fax: 886-2-23825962.CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.26121Potential conflict of interest: Dr. Yu consults for and received grants from Abbott. He is on the speakers’ bureau of and received grants from Roche. He also

received grants from Bristol-Myers Squibb. Dr. Chen consults for, advises, is on the speakers’ bureau of, and received grants from Bristol-Myers Squibb. He consultsfor Roche and advises Abbott.Additional Supporting Information may be found in the online version of this article.

HEPATOLOGY, Vol. 57, No. 3, 2013 YANG ET AL. 935

University Hospital.23 A detailed description of thisstudy has been published.23 Briefly, all patientsreceived 5 million units (MU) IFN-a-2b daily for 4weeks and then 5MU IFN-a-2b thrice a week foranother 28 weeks with and without ribavirin. Theremaining 161 patients received pegylated IFN-a-2a180 lg weekly for 24 or 48 weeks. Among these 161patients, 112 were consecutively enrolled in a studyinvestigating host and viral factors in HBeAg-positiveCHB patients receiving the treatment with pegylatedIFN-a-2a between December 2005 and December2008.20 The other 49 patients were consecutively en-rolled at the China Medical University Hospital fromNov 2004 to May 2009. All serum samples werestored at �80� until used. All enrolled patients signedinformed consent. This study was approved by theEthical Committee of the National Taiwan UniversityHospital.In Taiwan, the Bureau of National Health Insurance

launched a reimbursement program for treatment ofCHB since October 2003. According to the Asian-Pa-cific consensus statement on the management ofCHB,24 the HBeAg-positive CHB patients with pre-treatment serum ALT levels over two times the ULNwere eligible for treatment with pegylated IFN-a-2afor 24 weeks. Therefore, most of our patients receivingpegylated IFN-a-2a were treated for 24 weeks, andonly 30 patients received 48 weeks of pegylated IFN-a-2a.Definition of Treatment Responses. HBeAg sero-

clearance was defined by the loss of HBeAg in anHBeAg-positive CHB patient.15 The primary thera-peutic endpoint was HBeAg seroclearance or serocon-version at 24 weeks posttherapy. The secondary thera-peutic endpoint was HBeAg seroclearance orseroconversion with HBV DNA < 2,000 IU/mL and20,000 IU/mL at 24 weeks posttherapy.HBV DNA Extraction. HBV viral DNA was

extracted from 200 lL frozen serum samples using theQIAamp DNA Blood Mini kit (Qiagen, Germany)and eluted into 50 lL AE buffer according to themanufacturer’s instructions.Quantification of the Percentage of PC and BCP

Mutants by PCR Pyrosequencing. The detailed proce-dures are described in the Supporting Material. Briefly,the regions encoding the PC (nt1896) and BCP(nt1762 and nt1764) mutation sites were amplified bypolymerase chain reaction (PCR) with three primers, a50-biotinylated universal M13 primers (5b-M13) and apair of amplification primers, including the pairs foreither PC mutation (F1825 and R1931-M13) or BCPmutation (F1676-M13 and R1819) (Supporting Table

1). The primers were mixed at a ratio of 10 (theprimer without M13 tag) to 9 (5b-M13) to 1 (theprimer with M13 tag)25 to generate the M13-taggedbiotinylated-PCR product for subsequent single-stranded DNA (ssDNA) purification and pyrosequenc-ing. For the pyrosequencing reaction, we first isolatedbiotin-labeled ssDNA with the PyroMark VacuumPrep Workstation (Qiagen, Germany) by following themanufacturer’s protocol. The beads capturing ssDNAwere released into a PSQ HS 96 plate prefilled withthe sequencing primer (F1875-seq for the PC muta-tion and R1783-seq for the BCP mutation) (Support-ing Table 1) in the annealing buffer. Only the immo-bilized strand was used for pyrosequencing. Real-timepyrosequencing of the immobilized strand was per-formed with up to 96 samples in parallel at 28�C withthe PyroMark Q96 ID Instrument (Qiagen, Germany)using 96 PyroMark Gold Q96 reagents, including theenzyme and substrate mixtures, following the manufac-turer’s instructions. All the samples were independentlymeasured at least twice and the average of PC andBCP mutant percentages were calculated.Statistical Analysis. All factors collected are pre-

sented as the means and standard deviations (SDs) forcontinuous variables and frequency distribution forcategorical variables. Unconditional logistic regressionwas conducted to estimate the odds ratios (OR) and95% confidence intervals (CI) for the associationbetween PC and BCP mutant percentages and thechance of HBeAg seroclearance/seroconversion. Among91 subjects with more than one measurement of PCand BCP mutant percentages, the slopes of theirchanges over the five timepoints were estimated by thegeneral linear regression model. Using these slopes foreach individual as an independent variable, we con-ducted further unconditional logistic regression modelto estimate the association between these slopes ofchange over time and HBeAg seroclearance/seroconversion.

ResultsDevelopment and Validation of a Novel Pyrose-

quencing Assay for Quantification of PC and BCPMutations. The DNA template was amplified by PCRwith a pair of amplification primers, one of whichcontained M13-tagged sequence, and a 50-biotinylateduniversal M13 primer. The resulting PCR product wassubjected to ssDNA purification and subsequent pyro-sequencing. The detailed procedures are described inthe Materials and Methods and the Supporting Mate-rial. The primer design and PCR amplification strategy

936 YANG ET AL. HEPATOLOGY, March 2013

are illustrated in Fig. 1A. To validate the accuracy ofthis assay, we generated four reference plasmids con-taining wildtype PC, mutant PC, wildtype BCP, andmutant BCP sequences, respectively, which serve asstandards. We then mixed the wildtype and mutantPC and BCP plasmids with a range of predefinedratios, from 0% to 100% mutant in increments of10%. Our data showed that the measured percentagesof PC and BCP mutants were quite similar to the pre-defined ratios (Fig. 1B,C). The standard error of tripli-cate measurement of an individual sample was within5% (mostly, within 3%). We concluded that this assaycould achieve high accuracy with R2 of 0.99 within adynamic range between 10% and 90%. Therefore, we

further took advantage of this new assay to analyze thepercentages of PC and BCP mutants in the serial sam-ples of 203 HBeAg-positive CHB patients whoreceived IFN-based therapy. All the samples weremeasured twice using the same assay, and the resultsshowed high reproducibility for both PC and BCPmutation percentages (the correlation coefficient r ¼0.99, data not shown).Baseline Characteristics of Patients. The demo-

graphic data of the 203 HBeAg-positive CHB patientsare shown in Table 1. Among them, there were 42patients receiving 32 weeks of standard IFN with (21patients) or without ribavirin (21 patients) and theremaining 161 patients receiving pegylated IFN for 24

Fig. 1. Illustration and validation ofthe quantitative PCR pyrosequencingassay for the PC and BCP mutations.(A) Schematic illustration of the three-primer strategy for amplification of pre-core-encoding region and the resultant50-biotinylated and M13-tagged PCRproduct for the subsequent ssDNA purifi-cation. (B,C) Validation of the quantita-tive PCR-pyrosequencing assay for (B)PC and (C) BCP mutations using thereference plasmids with a predefinedmutant percentage from 0 to 100%.Each sample was measured independ-ently in triplicate and the data areshown as mean 6 SE.


weeks (131 patients) or 48 weeks (30 patients). Theprevious study has reported that adding ribavirin doesnot increase the efficacy of IFN on HBeAg seroconver-sion.23 The percentages of PC and BCP mutation rep-resented the frequencies of these two mutants amongthe serum HBV population within a single individual.In these IFN-treated cohorts, there were in total 55patients and 64 patients achieved HBeAg seroconver-sion and HBeAg seroclearance, respectively, at 24weeks post-IFN therapy.Predictive Value of Pretreatment PC and BCP

Mutant Percentages in HBeAg Seroconversion ofCHB Patients Receiving IFN Therapy. To determinethe role of PC and BCP mutant percentages in IFN-induced HBeAg seroconversion, we analyzed themusing this new quantitative PCR pyrosequencing assay.We measured the PC and BCP mutant percentages atdifferent timepoints from pretreatment to 24 weeks af-ter treatment. The comparisons of the mutant percen-tages of PC, BCP (A1762T), and BCP (G1764A) atbaseline (pretreatment) between the patients with andwithout IFN-induced HBeAg seroconversion were

33.1 6 30.8 versus 18.5 6 26.6, 39. 1 6 37.2 versus29.4 6 35.4, and 40.5 6 42.8 versus 28.5 6 40.2,respectively (Table 2). Using univariate logistic regres-sion analysis, we found that the PC G1896A mutationat baseline was significantly associated with HBeAgseroconversion (Table 2). The association between theBCP mutation G1764A at baseline and HBeAg sero-conversion was only borderline significant (P ¼0.079). The chance of HBeAg seroconversion wasincreased by 1.7% (OR ¼ 1.017, 95% CI: 1.007-1.028, P ¼ 0.002) per 1% increase of PC mutant fre-quency at baseline. Using HBeAg seroclearance as thetherapeutic endpoint, we found that, similarly, thechance of HBeAg seroclearance was significantly asso-ciated with both PC and BCP mutant frequencies(G1764A) at baseline (Table S2). However, whenHBeAg seroconversion with low HBV DNA (<2000IU/mL) was used as the therapeutic endpoint, onlyPC mutation at baseline (OR ¼ 1.026, 95% CI:1.013-1.040, P < 0.001), but not BCP mutations,was significantly associated with the therapeuticresponse.

Table 1. Baseline Characteristics of 203 HBeAg-Positive CHB Patients Receiving IFN-Based Therapy

IFN (n ¼ 21)

IFNþRBV

(n ¼ 21)

PegIFN (24wk)

(n ¼ 131)

PegIFN (48wk)

(n ¼ 30)

Total

(n ¼ 203)

Gender (M/F) 20/1 17/4 92/39 20/10 149/54

Age (yr) 29.1 6 5.1 30.3 6 7.7 34.8 6 9.4 35.1 6 8.8 33.8 6 9.0

ALT (U/mL) 200.8 6 91.6 191.6 6 78.8 213.5 6 147.8 152.0 6 103.1 94.1 6 167.6

Log-HBV-DNA (cps/mL) 8.01 6 0.89 8.26 6 0.88 7.55 6 1.63 8.10 6 1.49 7.80 6 1.51

Genotype (B/C/unknown) 10/10/1 15/6/0 85/46/0 25/5/0 135/67/1

PC G1896A (%) 12.6 6 20.6 27.2 6 30.4 23.9 6 30.4 22.9 6 25.0 22.9 6 28.9

BCP A1762T (%) 30.8 6 36.8 21.2 6 33.5 33.8 6 36.4 34.4 6 35.4 31.9 6 36.0

BCP G1764A (%) 32.0 6 41.8 20.2 6 37.4 33.0 6 41.6 35.5 6 41.8 31.6 6 41.1

HBeAg seroconversion (Yes/No) 5/16 5/16 33/98 12/18 55/148

HBeAg seroconversion with HBV

DNA < 2,000 IU/mL (Yes/No)

2/19 2/19 20/111 5/25 29/174

HBeAg seroconversion with HBV

DNA < 20,000 IU/mL (Yes/No)

3/18 4/17 22/109 7/23 36/167

HBeAg loss (Yes/No) 6/15 6/15 40/91 12/18 64/139

HBeAg loss with HBV DNA < 2,000 IU/mL (Yes/No) 3/18 3/18 23/108 5/25 34/169

HBeAg loss with HBV DNA < 20,000 IU/mL (Yes/No) 4/17 5/16 26/105 7/23 42/161

Table 2. Univariate Analysis of the Association Between PC and BCP Mutant Percentages and HBeAg Seroconversion(left)/HBeAg Seroconversion With HBV DNA < 2000 IU/mL (right) at 6 Months Off Therapy

HBeAg Seroconversion HBeAg Seroconversion with HBV DNA < 2000 IU/mL

% of Mutant (mean 6 S.D)

Odds Ratio (95% CI) P

% of Mutant (mean 6 S.D)

Odds Ratio (95% CI) PResponder Nonresponder Responder Nonresponder

Baseline (per 1%

increase)*

PC G1896A 33.1 6 30.8 18.5 6 26.6 1.017 (1.007-1.028) 0.002 44.0 6 34.2 19.3 6 26.2 1.026 (1.013-1.040) <0.001

BCP A1762T 39.1 6 37.2 29.4 6 35.4 1.007 (0.999-1.016) 0.104 33.7 6 35.8 31.3 6 36.1 1.002 (0.990-1.014) 0.754

BCP G1764A 40.5 6 42.8 28.5 6 40.2 1.007 (0.999-1.015) 0.079 33.8 6 41.4 31.0 6 41.0 1.002 (0.991-1.012) 0.753

*The odds ratio of each individual mutant at baseline represents the odds ratio of HBeAg seroconversion and HBeAg seroconversion with HBV DNA <2000 IU/

mL per 1% increment of the indicated mutant percentage at baseline.


We further took into consideration other baselinehost and viral factors, including age, gender, ALT level,HBV viral load, and HBV genotype. Previous studieshave shown that the dual mutations, A1762T andG1764A, of BCP mutants are highly associated, andour own data also showed the high correlation (r ¼0.991) between these two BCP mutations among allthe samples we measured Supporting Fig. 1. We thustook either A1762T or G1764A mutation as an indica-tor of BCP mutation for the multivariate analysis.Actually, the results using either mutation for calcula-tion came to the same conclusion (data not shown).Therefore, without specific mention, the percentage ofA1762T was used for the following analysis regardingBCP mutation. Using multivariate logistic regressionanalysis we further showed that, at baseline, higherpretreatment PC and BCP mutant percentages wereindependently associated with higher rate of HBeAgseroconversion (Table 3). The chance of HBeAg sero-conversion was increased by 2.2% (OR ¼ 1.022, 95%CI: 1.009-1.034, P ¼ 0.001) and 2.3% (OR ¼ 1.023,95% CI:1.010-1.037, P ¼ 0.001) per 1% increase ofPC and BCP mutants, respectively. We also demon-strated the similar effects of PC and BCP mutant per-centages on the treatment response when HBeAg sero-clearance was adopted as a therapeutic endpoint (TableS3). However, using the HBeAg seroconversion withlow HBV DNA (<2,000 IU/mL) as the therapeuticendpoint, we demonstrated that only the PC mutationpercentage was significantly associated with the treat-

ment response (OR ¼ 1.030, 95% CI: 1.014-1.047,P < 0.001) (Table 3).HBsAg loss is the ultimate therapeutic endpoint in

patients receiving IFN treatment. However, in thesecohorts we did not find any patients exhibiting HBsAgloss. To further investigate the relationship betweenthe percentages of PC and BCP mutants and HBsAgloss, we decided to adopt alternative endpoints, suchas HBsAg < 1,000 IU/mL at 24 weeks posttherapyand HBsAg decrease 1 or 2 logs during IFN treat-ment. However, we could not demonstrate the predic-tive role of PC and BCP mutation percentages in thesetherapeutic endpoints.Differential Evolution Patterns of PC and BCP

Mutants During HBeAg Seroconversion. To investi-gate the dynamic changes of PC and BCP mutantsduring IFN treatment, we carried out an extensiveanalysis of the percentages of PC and BCP mutationsin 91 patients (42 with IFN with/without ribavirinand 49 with 24 or 48 weeks of pegylated IFN) whohad serial serum samples from the start of treatmentto 24 weeks after the end of treatment. The evolutionpatterns of the PC and BCP mutants in patientsgrouped by HBeAg seroconversion with/without lowHBV DNA are illustrated using boxplots (Fig. 2). Wefound that PC mutation, but not BCP mutations,increased in patients with HBeAg seroconversion. Inaddition, compared to the patients with HBeAg sero-conversion and low HBV DNA (<2,000 IU/mL),those with HBeAg seroconversion and high HBV

Table 3. Multivariate Analysis of Factors Associated With HBeAg Seroconversion (left) and HBeAg Seroconversion With HBVDNA < 2000 IU/mL at 6 Months Off Therapy

HBeAg Seroconversion

HBeAg Seroconversion

with HBV DNA < 2000 IU/mL

Characteristics OR (95% Cl) P-value OR (95% Cl) P-value

Age ( per 1 year increase) 0. .970 (0.926-1.014) 0.184 0. 941 (0.884-1.003) 0.063

Gender 0.649 0.802

Male 1.00 1.00

Female 0. 825 (0.361-1.887) 1.151 (0.385-3.437)

ALT (per 1 U/L increase) 1.000 (0.998-1.003) 0.844 1.001 (0. 998-1.004) 0.500

Treatment group

IFN 1.00 1.00

IFNþRBV 0. 722 (0.158-3.302) 0.675 0. 629 (0.070-5.645) 0.679

PegIFN (24wk) 0.559 (0.163-1.911) 0.354 0. 919 (0.165-5.110) 0.923

PegIFN (48wk) 0.755 (0.170-3.349) 0.711 1.712 (0.237-12.38) 0.594

Genotype 0.004 0.520

B 1.00 1.00

C 0.192 (0. 062-0.592) 0.640 (0.164-2.494)

HBV titer (per 1 log10 HBV DNA increase) 1.011 (0.776-1.317) 0.937 0. 957 (0. 696-1.316) 0.787

Baseline (per 1% increase)*

PC G1896A mutant 1.022 (1.009-1.034) 0.001 1.030 (1.014-1.047) <0.001

BCP A1762T mutant 1.023 (1.010-1.037) 0.001 1.011 (0.994-1.029) 0.199

*The odds ratio of each individual mutant at baseline represents the odds ratio of HBeAg seroconversion and HBeAg seroconversion with HBV DNA < 2000 IU/

mL per 1% increment of the indicated mutant percentage at baseline.


DNA (>2,000 IU/mL) tended to have higher PCmutation frequency during treatment. We also usedHBeAg seroclearance for patient grouping and found

that the PC mutation, but not BCP mutation, accu-mulated in patients with HBeAg seroclearance alongthe course of IFN treatment Supporting Fig. 2.By using the slopes over time indicating the dynamic

changes of PC and BCP mutations, we further investi-gated the relationship between the rate of change of PCand BCP mutations and HBeAg seroconversion/sero-clearance with low HBV DNA. The results revealed thatthe slope of PC mutation, but not that of BCP mutation,was significantly associated with HBeAg seroclearancewith HBV DNA < 20,000 IU/mL (OR ¼ 8.897, 95%CI: 1.118-70.797, P ¼ 0.039), and was also marginallyassociated with HBeAg seroclearance with HBV DNA <2,000 IU/mL (OR ¼ 9.999, 95% CI: 0.713-140.277, P¼ 0.088) (Table 4). However, when HBeAg seroconver-sion was adopted as the endpoint, the slope of PC muta-tion was only marginally associated with HBeAg sero-conversion with HBV DNA < 20,000 IU/mL (OR ¼8.736, 95% CI: 0.677-112.818, P ¼ 0.097), but wasnot associated with HBeAg seroconversion with HBVDNA < 2,000 IU/mL (Table S4). This is probably dueto fewer patients having HBeAg seroconversion with lowHBV DNA. Taken together, these results indicated thatthe percentage of PC mutation, but not that of BCPmutation, did increase along the course of the IFN-basedtherapy in patients achieving HBeAg seroclearance.Association Between the PC Mutant Percentages

at the End of Treatment (EOT) and the Viral LoadAfter HBeAg Seroconversion. Although HBeAg sero-conversion is often accompanied by a favorable prog-nosis, HBeAg-negative hepatitis sometimes occurs afterHBeAg seroconversion. HBeAg-negative hepatitis is of-ten associated with higher HBV DNA levels.

Fig. 2. The evolution patterns of PC and BCP mutants along thecourse of IFN therapy. The distribution of PC (A) and BCP (B) mutantpercentages at different timepoints are illustrated using a boxplot.Patients were grouped based on occurrence of HBeAg seroconversionwith/without low HBV DNA (<2,000 IU/mL). The borders of each boxrepresents the 25 percentile (lower) and 75 percentile (upper) and theline within the box indicates the median mutant percentage. The topand bottom whiskers indicate the maximum and minimum of the data.

Table 4. Multivariate Analysis of Factors Associated With HBeAg Loss at 6 Months Off Therapy Based on the Slope of thePC and BCP Mutant Percentages Over Time

HBeAg Seroclearance with

HBV DNA < 2000 IU/mL

HBeAg Seroclearance with HBV

DNA < 20000 IU/mL

Characteristics OR (95% Cl) P-value OR (95% Cl) P-value

Age (pr 1 year increase) 0.995 (0.884-1.120) 0.936 0.953 (0.861-1.056) 0.358

Gender 0.143

Male 1.00

Female 5.486 (0.563-53.489)

ALT (U/L, c) 1.004 (0.993-1.014) 0.498 1.005 (0.996-1.014) 0.302

Treatment group

IFN 1.00 1.00

IFNþRBV 2.942 (0.327-26.488) 0.336 2.975 (0.458-19.338) 0.254

PegIFN (24wk) 2.834 (0.202-39.810) 0.440 1.335 (0.125-14.263) 0.811

PegIFN (48wk) 4.145 (0.300-57.347) 0.289 6.511 (0.698- 60.780) 0.100

Genotype 0.879 0.673

B 1.00 1.00

C 0.857 (0. 117-6.262) 0.705 (0.140-3.564)

HBV titer (per 1 log10 HBV DNA increase) 0.953 (0.419-2.170) 0.909 0.925 (0.443-1.931) 0.835

PC G1896A mutant percentage slopes over time 9.999 (0.713-140.277) 0.088 8.897 (1.118-70.797) 0.039

BCP A1762T mutant percentage slopes over time 3.438 (0.330-35.866) 0.302 1.619 (0.205-12.801) 0.648


Therefore, we investigated whether the percentages ofPC and BCP mutants affected the levels of viral repli-cation after HBeAg seroclearance. Among the 55patients who achieved HBeAg seroconversion, 28patients had stored sera for comprehensive analysis ofPC and BCP mutations at different timepoints. Inter-estingly, in these 28 patients with HBeAg seroconver-sion, a higher percentage of PC mutants at EOT wasassociated with a higher chance of having high HBVviral load after HBeAg seroconversion. Four out of six(66.7%) individuals with the highest PC mutants(>75% of PC mutation) at EOT exhibited high vire-mia (>200,000 IU/mL), whereas none of the 10patients with the lowest PC mutants (<25% of PCmutation) had a high viral load (Fig. 3). In addition,the PC mutant percentages at EOT in patients withhigh viremia after HBeAg seroconversion was signifi-cantly higher than those in patients without high vire-mia (73.7% versus 35.8%, P ¼ 0.002). Analyzing the35 patients who had HBeAg seroclearance and avail-able serum samples at EOT for analysis, we also foundthat the patients with a higher percentage of PC muta-tion at EOT were more likely to have high viremia af-ter HBeAg seroclearance Supporting Fig. 2.

Discussion

In this study we comprehensively analyzed the per-centages of PC and BCP mutations at baseline andduring and after IFN treatment in a large number ofHBeAg-positive CHB patients with HBV genotype Bor C infection. Our results clearly demonstrated thevalue of both PC and BCP mutation percentages at

baseline in predicting HBeAg seroconversion.Although previous studies suggested the association ofbaseline BCP mutation with IFN-induced HBeAgseroconversion,16,17,20 the role of pretreatment PCmutation in HBeAg seroconversion is somewhat con-troversial. Lok et al.18 showed that HBeAg-positiveCHB patients with PC mutation is more likely to clearHBeAg, but other studies failed to demonstrated thisassociation.16,17,19,20 These discrepant findings prob-ably resulted from the use of less sensitive and inform-ative qualitative assays, although the influence of thedifferent ethnic and geographic origins in these studiescannot be excluded. By quantitative analysis of PCand BCP mutations, we found that the percentages ofboth PC and BCP mutants at baseline could predictIFN-induced HBeAg seroconversion, suggesting theimportance and utility of the quantitative analysis ofHBV mutants in evaluating the prognosis of CHB.Interestingly, although both PC and BCP mutant

percentages at baseline were associated with HBeAgseroconversion, only the percentage of PC mutation,but not that of BCP mutation, was associated withHBeAg seroconversion plus low HBV DNA. Detailedanalysis of the viral evolution from baseline to 24weeks posttherapy confirmed the distinct evolutionpatterns of PC and BCP mutations during IFN-induced HBeAg seroconversion. Chu et al.11 consis-tently showed that the PC mutant ratio increased dur-ing spontaneous HBeAg seroconversion. Recently, Nieet al.12 also quantitatively analyzed the dynamicchanges of PC and BCP mutants in 13 CHB patientswho had spontaneous HBeAg seroconversion. Theyfound that PC and/or BCP mutant ratios increasedduring HBeAg seroconversion. However, differentfrom Nie et al.’s study, our results showed that the PCmutant percentage increased, but the BCP mutant per-centage was relatively unchanged during HBeAg sero-conversion. The reasons for the different observationsbetween Nie et al.’s study and ours remain unclear,although they might be due to the difference betweenspontaneous and IFN-induced HBeAg seroconversion,or due to sampling bias between studies.The distinct evolution patterns of PC and BCP

mutations during HBeAg seroconversion might suggestdifferent biological mechanisms involved in selectionof PC and BCP mutants. Consistently, a prior long-term longitudinal follow-up study on a large CHBcohort revealed that BCP mutation enhances the devel-opment of HCC, but PC mutation is associated withthe decreased risk of HCC,14 indicating the distinctbiological effects of PC and BCP mutations on thedisease status of CHB. It has been suggested that

Fig. 3. The association between the PC mutant percentage at EOTand the probability of high viremia after HBeAg seroconversion. Thepatients with IFN-induced HBeAg seroconversion were divided into fourgroups according to the levels of PC mutant percentage at EOT. Thenumbers shown above the bar graph indicate the number of thepatients with high viremia (HBV DNA level >200,000 IU/mL) over thetotal number of patients in each group with the indicated level of PCmutation.


cytosolic HBeAg is an immunogen targeted by thehost immune response for elimination of infected he-patocytes during the immune clearance phase.26 Alongthe course of HBeAg seroclearance, the host immunepressure tends to select HBeAg-negative viral strains,like PC mutants, probably through immune-mediatedhepatocytolysis. This might explain the accumulationof PC mutation during HBeAg seroconversion. How-ever, the percentage of BCP mutation at baseline wasalso associated with HBeAg seroconversion, although itwas not associated with HBeAg seroconversion pluslow HBV DNA level and did not significantly increaseduring HBeAg seroconversion. This fact implies thatthe percentage of BCP mutant probably reflects thestrength of host immune response against HBV, butthis immune pressure is not the direct driving force tocause HBeAg seroconversion. Therefore, the accumula-tion of BCP mutants may not be observed during ashort time frame in which HBeAg seroconversionoccurs, but can be seen in a long period of follow-up,like in Nie et al.’s study.12 It would be interesting toexamine this phenomenon in patients undergoingspontaneous HBeAg seroconversion and correlate thequantities of PC or BCP mutants with their long-termclinical outcomes.Interestingly, in contrast to the above observation

that a higher pretreatment PC mutant percentage andits increase during IFN treatment had a favorable out-come, HBeAg seroconversion, in the HBeAg-positivepatients receiving IFN therapy, we found a high per-centage of PC mutant at the end of IFN therapy seemedto be associated with high viremia after HBeAg serocon-version. Consistently, several previous studies demon-strated that PC mutant prevailed in patients withHBeAg-negative hepatitis.27,28 In addition, by quantita-tive analysis of the PC mutant ratios, Chu et al.11

reported that all patients with high viremia and ALTelevation after HBeAg seroconversion exclusively exhib-ited the PC mutant. Although the underlying mecha-nisms are not known, we speculated that a very highpercentage of PC mutation at EOT probably impliesthe efficient escape of viral mutants from the immunecontrol, and the failure of the host immune response tocontain viral replication. Alternatively, a high percent-age of PC mutation may be associated with some otherHBV mutations that cause high viremia. Because only28 patients with HBeAg seroconversion had availablesera at EOT for analysis in this study, a larger cohortwith longitudinal and comprehensive analysis of theentire HBV genome may be required to solve this issue.One drawback of this study was the heterogeneous

treatment populations, which might compromise our

conclusion to some extent. Because the reimbursementpolicy of the Bureau of National Insurance in Taiwan,most of the patients receiving pegylated IFN-a-2a inthis cohort were treated for 24 weeks, not for 48weeks. The latter is the current preferred regimendemonstrated by the NEPTUNE study.29 Therefore,further studies are required to validate our observationin the HBeAg-positive CHB patients receiving 48weeks of IFN-a-2a.In summary, by detailed quantitative analysis of lon-

gitudinal samples from a large number of HBeAg-posi-tive CHB patients receiving IFN therapy, we havedemonstrated different predictive values of PC andBCP mutants in HBeAg seroconversion. Particularly,our data revealed distinct and previously unnoticedevolution patterns of PC and BCP mutants duringIFN-induced HBeAg seroconversion. Understandingthe underlying mechanisms that regulate the dynamicevolution of PC and BCP mutants can help predictthe prognosis of CHB patients as well as design betterstrategies of follow-up and antiviral therapy for them.

Acknowledgment: We thank Drs. Hwei-Fang Tien,Wen-Chien Chou as well as Ming-Cheng Lee for helpin pyrosequencing.

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Distinct evolution and predictive value of hepatitis B virus precore and basal core promoter mutations in interferon-induced hepatitis B e antigen seroconversion