Figures
Abstract
Background and Aims
There is lack of a practical biomarker to predict sustained virological response (SVR) in chronic hepatitis B (CHB) patients undergoing peginterferon alfa-2a (PEG-IFN). The aim of this pilot study was to identify immunological features associated with SVR.
Methods
Consecutive 74 CHB patients receiving 24 weeks (for hepatitis B e antigen (HBeAg)-positive) or 48 weeks (for HBeAg-negative) PEG-IFN, were prospectively enrolled. Serum HBV viral loads, hepatitis B surface antigen (HBsAg), CXCL9, IFN-γ-inducible protein 10 (IP-10), interferon-gamma (IFN-γ) and transforming growth factor beta (TGF-β) were measured at baseline and week 12. SVR was defined as HBeAg seroconversion combined with viral load <2000 IU/mL in HBeAg-positive (n=36), and viral load <2000 IU/mL in HBeAg-negative patients (n=38) at 48 weeks after the end of treatment.
Results
Nineteen patients (25.7%), 7 in HBeAg-positive and 12 in HBeAg-negative, achieved SVR. There were significant declines of HBV DNA, HBsAg, IP-10 and IFN-γ levels at week 12. In multivariate analysis, pre-treatment CXCL9 >80 pg/mL, HBV DNA <2.5 x 107 IU/mL and on-treatment HBV viral load, HBsAg decline >10% at week 12 were predictors of SVR. The performance of CXCL9 in predicting SVR was good in patients with HBV DNA <2.5 x 107 IU/mL, particularly in HBeAg-negative CHB cases (positive predictive value, PPV= 64.3%).
Citation: Lee I-C, Huang Y-H, Su C-W, Wang Y-J, Huo T-I, Lee K-C, et al. (2013) CXCL9 Associated with Sustained Virological Response in Chronic Hepatitis B Patients Receiving Peginterferon Alfa-2a Therapy: A Pilot Study. PLoS ONE 8(10): e76798. https://doi.org/10.1371/journal.pone.0076798
Editor: Hans Tillmann, Duke University, United States of America
Received: April 19, 2013; Accepted: August 28, 2013; Published: October 4, 2013
Copyright: © 2013 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by grants from the National Science Council (NSC 100-2314-B-010-024), and Taipei Veterans General Hospitals (VGH100C-104, VGH101C-147, VGH102C-136), Taipei, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Chronic hepatitis B virus (HBV) infection remains a challenging global health problem, with more than 350 million carriers worldwide [1]. The spectrum and the natural course of chronic HBV infection are diverse and variable, ranging from an inactive carrier state to progressive chronic hepatitis B (CHB), which may evolve to hepatic decompensation, cirrhosis and hepatocellular carcinoma (HCC) [2]. Both host and viral factors play critical roles in the natural history of CHB, disease activity as well as efficacies of antiviral therapies [3,4]. Impaired host immune response against HBV is commonly observed in patients with CHB, which might be related to persistent high viral loads with subsequent T cell exhaustion [5]. Current antiviral strategies for CHB aim at effective viral suppression as well as restoration of HBV-specific immune responses.
Peginterferon-alpha-2a (PEG-IFN), which has direct antiviral and immuno-modulatory effects, is currently one of the first-line treatment options for CHB [6,7]. Antiviral therapy with PEG-IFN has been shown to be effective in suppressing HBV replication, and may result in hepatitis B e antigen (HBeAg) seroconversion, hepatitis B surface antigen (HBsAg) clearance, normalization of alanine aminotransferase (ALT) levels, and histological improvement [6,7]. Patients who achieve an IFN-induced virological response may have a long-term therapeutic effect and a reduced risk of cirrhosis and HCC [8,9]. However, only about 30% of CHB patients treated with PEG-IFN could achieve a sustained virological response (SVR) [10–13]. Several baseline and on-treatment indicators have been identified to predict treatment response to PEG-IFN [7,10]. In HBeAg-positive CHB, low viral load, high serum ALT levels, HBV genotype and high activity scores on liver biopsy are pre-treatment predictors of HBeAg seroconversion [14,15], whereas currently there are no strong pre-treatment predictors of virological response in HBeAg-negative CHB patients [7]. During the treatment, declines of viral load and HBsAg levels at 12 weeks are strong predictors of virological response in both HBeAg-positive and HBeAg-negative patients [16–18].
Based on the immuno-modulatory properties of PEG-IFN, host immune status may have influence on the efficacy of PEG-IFN for CHB. In recent years, several studies have shown the potential roles of cytokines and chemokines in chronic viral hepatitis. CXCL9 (monokine induced by IFN-γ [MIG]) and IP-10 (IFN-γ-inducible protein 10, also called CXCL10) have been reported to play important roles during hepatitis flares in CHB [19]. In patients with hepatitis C virus (HCV) infection, lower baseline and on-treatment IP-10 levels may predict a higher rate of virological response to IFN-based therapy [20–22]. Recently, we also demonstrated a correlation of IP-10 with higher hepatitis activity in patients with CHB [4]. Pro-inflammatory cytokines interferon-gamma (IFN-γ) and transforming growth factor beta (TGF-β) may also have roles in suppressing HBV replication [1,23,24]. However, the clinical significance of these cytokines and chemokines during PEG-IFN therapy is unclear. IL28B single nucleotide polymorphisms (SNPs) have been shown to correlate with IFN-induced treatment response in patients with chronic hepatitis C, as well as HBeAg seroconversion in HBeAg-positive CHB patients [25–28]. However, the role of IL28B SNPs in predicting SVR to PEG-IFN therapy for CHB patients is still debated.
Currently, pre-treatment and on-treatment predictors of SVR in CHB patients under PEG-IFN therapy remain unsatisfactory to meet clinical need. The aim of this pilot study was to investigate the dynamics of cytokines and chemokines during PEG-IFN treatment, and to evaluate the baseline and on-treatment immunological and viral factors associated with SVR in CHB patients treated with PEG-IFN.
Materials and Methods
Patients
From January 2010 to July 2011, consecutive 74 CHB patients receiving PEG-IFN alfa-2a 180µg per week at the Taipei Veterans General Hospital were prospectively enrolled. All patients were positive for serum HBsAg for more than 6 months and patients receiving PEG-IFN therapy fulfilled the treatment criteria for CHB according to the American Association for the Study of Liver Disease (AASLD) treatment guidelines, i.e. serum ALT levels >80 U/L [2 x upper limit of normal (ULN)] with HBV DNA >20,000 IU/mL in HBeAg-positive patients or >2,000 IU/mL in HBeAg-negative patients [6]. All patients were negative for any of the following points: (1) coinfection with HCV, hepatitis D virus, or human immunodeficiency virus, (2) alcoholic liver disease, (3) suspected autoimmune diseases with antinuclear antibody (ANA) titer ≥1:160, positive test for anti-smooth muscle antibody or anti-mitochondrial antibody, (4) use of hepatotoxic drug or Chinese herb, and (5) radiological evidence of cirrhosis or HCC (i.e., abdominal sonogram, computed tomography scan, or magnetic resonance imaging scans). In general, the treatment duration was 24 weeks for HBeAg-positive, and 48 weeks for HBeAg-negative cases, which is under the regulation of Bureau of National Health Insurance, Taiwan. In HBeAg-positive patients, additional 24 weeks of PEG-IFN was allowed without reimbursement.
After initiating PEG-IFN treatment, patients were subsequently followed at the outpatient clinic every 2 to 4 weeks. Peripheral blood samples and HBV viral loads were evaluated at baseline, week 12, the end of treatment (EOT), 24 and 48 weeks after PEG-IFN treatment for serological and virological tests. This study was approved by the Institutional Review Board, Taipei Veterans General Hospital, which complied with standards of the Declaration of Helsinki and current ethical guidelines. All patients provided written informed consents for participation of the study.
End point and definition
The primary end point was sustained virological response (SVR). In HBeAg-positive patients, SVR was defined as a combination of HBeAg seroconversion, indicated as seronegative of HBeAg and seropositive of anti-HBe in serum, with HBV DNA level less than 2000 IU/mL at 48 weeks after the end of PEG-IFN therapy. In HBeAg-negative patients, SVR was defined as HBV DNA level less than 2000 IU/mL at 48 weeks after the end of PEG-IFN therapy [7].
Liver biochemistry and viral serology tests
Serum biochemical studies were performed using a systemic multi-autoanalyzer (Technicon SMAC, Technicon Instruments Corp., Tarrytown, NY). The serum samples were tested for the presence of HBeAg and anti-HBe antibody using radio-immunoassay (Abott Laboratories, North Chicago, IL), while HBV DNA was determined by Roche Cobas Tagman HBV DNA assay (detection limit of 12 IU/mL, Roche Diagnostics, Switzerland).
Detection, genotyping and sequencing of HBV DNA
Genotyping of HBV was performed by PCR restriction fragment length polymorphism (PCR-RFLP) of the surface gene of HBV [29,30]. Briefly, DNA was extracted from serum, and the fragment of the HBV genome between nucleotide position 120 and 604 was amplified by semi-nested PCR. The PCR products were subsequently treated with restriction enzymes. After incubation, the samples were run on a 4% agarose gel and stained by ethidium bromide. To confirm the correct genotyping, direct sequencing from the PCR products was done.
To detect precore G1896A and basal core promoter (BCP) A1762T/G1764A mutations, sequencing of the core region of HBV DNA was performed in all patients. Semi-nested PCR was performed by using a pair of primers: internal primers 1653F (5’-CATAAGAGGACTCTTGGACT-3’, position 1653-1672) and 1974R (5’-GGAAAGAAGTCAGAAGGC-3’, position 1974-1957); external primers: 1623F (5’-TCGCATGGAGACCACCGTCT-3’, position 1623-1640) and 2076R (5’-ATAGCTTGCCTGAGTGC-3’, position 2076-2060) as previously described [6,21].
The PCR products were then subjected to the dye-terminator cycle sequencing reaction using specific primers according to the standard protocol provided by the manufacturer (Dye terminator cycle sequencing core kit no. 402117, Perkin Elmer Cetus Corp., Norwalk, CT). To avoid false positive results, instructions to prevent cross contaminations were strictly followed.
Serum HBsAg quantification
HBsAg levels were quantified using the Elecsys HBsAg II assay (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. The detection limit of the Elecsys HBsAg II assay was 0.05 IU/mL.
Enzyme-linked sorbent assay (ELISA)
The concentrations of CXCL9 and IP-10 were tested by commercialized human cytokine ELISA kits from PeproTech (Rocky Hill, NJ). The concentrations of IFN-γ and TGF-β were tested by commercialized human cytokine ELISA kits from eBioscience (San Diego, CA). The procedure followed the instruction provided by manufactures.
IL28B genotyping
Four SNPs of IL28B including rs8105790, rs12979860, rs8099917 and rs10853728 were chosen according to previous reports and our study [4,25–28,31]. The genotype of rs12979860 was tested using TaqMan custom-designed rs12979860 probes (Applied Biosystems, Foster City, CA; forward primer GCCTGTCGTGTACTGAACCA, reverse primer GCGCGGAGTGCAATTCAAC, and the probes TGGTTCGCGCCTTC [VIC] and CTGGTTCACGCCTTC [FAM], respectively) [32]. The genotypes of the rs8105790, rs8099917 and rs10853728 were determined with the ABI TaqMan SNP genotyping assays (Applied Biosystems) and with predesigned commercial genotyping assays (ABI assay C__43813808_10, C__11710096_10, C__11710090_10). Briefly, PCR primers and two allelic-specific probes was designed to detect a specific SNP target. The PCR reactions were performed in 96-well microplates with ABI 7900 real-time PCR (Applied Biosystems) as previously described [4].
Statistical analyses
All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS 17.0 for Windows, SPSS Inc, Chicago, IL). Values were expressed as median (ranges) or as mean ± standard deviation when appropriate. The correlation between serum ALT and chemokine levels were tested by Spearman’s test. Pearson chi-square analysis or Fisher exact test was used to compare categorical variables, while the Student t test or Mann-Whitney U test was used to compare continuous variables. Wilcoxon signed ranks test was used to compare serial changes in virological and immunological factors. Variables with p<0.1 were analyzed by multivariate logistic regression analysis to identify independent variables for predicting SVR. The best cut-off value of each variable was determined by the receiver-operating characteristic (ROC) curve (Youden index) using MedCalc (version 4.20, MedCalc Software, Mariakerke, Belgium). A 2-tailed p value <0.05 was considered statistically significant.
Results
Characteristics of CHB patients receiving PEG-IFN therapy
Baseline characteristics of the 74 CHB patients were summarized in Table 1. Patients were predominantly males (77%) and genotype B (64.9%). BCP mutations and precore mutations were observed in 31.1% and 47.3% of patients, respectively. The prevalence of the major IL28B genotypes rs8105790 TT, rs12979860 CC, rs8099917 TT, and rs10853728 CC were 94.6%, 93.2%, 94.6% and 71.6%, respectively. In the 36 HBeAg-positive patients, 30 (83.3%) received 24-weeks and 5 6 (16.7%) received 48-week PEG-IFN therapy. All 38 HBeAg-negative patients received 48-week PEG-IFN therapy. Seventy-three (98.6%) patients completed the full course of PEG-IFN therapy, while 1 (1.4%) patient discontinued PEG-IFN treatment due to intolerance and were switched to oral antiviral therapies. The patient who failed to complete PEG-IFN therapy was classified as non-responder to PEG-IFN for analysis in this study.
| All patients n = 74 | No SVR n = 55 (74.3%) | SVR n = 19 (25.7%) | p | |
|---|---|---|---|---|
| Age (years) | 41.6 ± 10.2 | 40.5 ± 10.0 | 44 ± 11 | 0.119 |
| Male sex, n (%) | 57 (77.0) | 42 (76.4) | 15 (78.9) | 1.000 |
| Treatment duration | 0.514 | |||
| 24 weeks | 30 (40.5) | 24 (43.6) | 6 (31.6) | |
| 48 weeks | 44 (59.5) | 31 (56.4) | 13 (68.4) | |
| HBeAg-positive, n (%) | 36 (48.6) | 29 (52.7) | 7 (36.8) | 0.353 |
| Genotype | 0.922 | |||
| B, n (%) | 48 (64.9) | 35 (63.6) | 13 (68.4) | |
| C, n (%) | 26 (35.1) | 20 (36.4) | 6 (31.6) | |
| BCP mutation, n (%) | 23 (31.1) | 18 (32.7) | 5 (26.3) | 0.816 |
| Precore mutation, n (%) | 35 (47.3) | 23 (41.8) | 12 (63.2) | 0.180 |
| IL28B polymorphisms | ||||
| rs8105790 TT/CT/CC, n (%) | 70/4/0 (94.6/5.4/0) | 51/4/0 (92.7/7.3/0) | 19/0/0 (100/0/0) | 0.353 |
| rs12979860 CC/CT/TT, n (%) | 69/5/0 (93.2/6.8/0) | 50/5/0 (90.9/9.1/0) | 19/0/0 (100/0/0) | 0.319 |
| rs8099917 TT/GT/GG, n (%) | 70/4/0 (94.6/5.4/0) | 51/4/0 (92.7/7.3/0) | 19/0/0 (100/0/0) | 0.567 |
| rs10853728 CC/CG/GG, n (%) | 53/20/1 (71.6/27/1.4) | 39/15/1 (70.9/27.3/1.8) | 14/5/0 (73.7/26.3/0) | 1.000 |
| ALT (U/L) | 216 ± 200 | 202 ± 180 | 258 ± 251 | 0.276 |
| AST (U/L) | 110 ± 98 | 99 ± 78 | 144 ± 139 | 0.205 |
| HBV DNA (Log10 IU/mL) | 7.16 (3.36-8.04) | 7.34 (3.55-8.04) | 5.82 (3.36-8.04) | 0.004 |
| HBsAg (Log10 IU/mL) | 3.49 (1.45-5.46) | 3.64 (1.53-5.46) | 3.14 (1.45-5.27) | 0.028 |
| CXCL9 (pg/mL) | 65.1 (12.3-1000) | 45.3 (12.3-1000) | 191.7 (22.0-1000) | 0.002 |
| IP-10 (pg/mL) | 89.6 ± 56.8 | 101.7 ± 55.2 | 106.1 ± 62.7 | 0.833 |
| IFN-γ (pg/mL) | 30.2 (undetectable-1416) | 32.7 (undetectable-1416) | 29.6 (undetectable-886) | 0.420 |
| TGF-β (pg/mL) | 939 ± 503 | 903 ± 537 | 1040 ± 388 | 0.232 |
Table 1. Baseline characteristics of the CHB patients with or without sustained virological response (SVR).
Treatment response
Overall, 19 patients (25.7%) had achieved SVR at 48 weeks after the end of PEG-IFN treatment. Among the 36 HBeAg-positive patients, 12 (33.3%) achieved HBeAg seroconversion, including 10 of the 30 patients (33.3%) receiving 24-week course of treatment and 2 of the 6 patients (33.3%) receiving 48-week course of treatment; whereas only 7 (19.4%) finally achieved SVR, including 6 of the 30 patients (20%) receiving 24-week course of treatment and 1 of the 6 patients (16.7%) receiving 48-week course of treatment. In HBeAg-negative patients, 12 (31.6%) achieved SVR and 2 cases (5.3%) achieved HBsAg clearance at 48 weeks after the end of PEG-IFN treatment. During the extended follow-up period between 24 to 48 weeks post treatment, one HBeAg-positive patient developed HBeAg seroconversion and one HBeAg-negative patient had viral relapse.
Dynamic changes of virological and immunological factors
Dynamic changes of serum HBV DNA, HBsAg, cytokine and chemokine levels from baseline to week 12 of PEG-IFN treatment were shown in Table 2. Viral load, HBsAg, IP-10 and IFN-γ levels significant declined after 12 weeks of PEG-IFN treatment. No significant changes were observed in CXCL9 and TGF-β levels from baseline to week 12 in overall patients.
| Baseline | Week 12 | p* | |
|---|---|---|---|
| HBV DNA, Log10 IU/mL | 7.16 (3.36-8.04) | 4.12 (undetectable -8.04) | <0.001 |
| HBsAg, Log10 IU/mL | 3.49 (1.45-5.46) | 3.43 (0.89-4.93) | <0.001 |
| CXCL9, pg/mL | 65.1 (12.3-1000) | 52.1 (1.7-1000) | 0.147 |
| IP-10, pg/mL | 89.6 ± 56.8 | 73.4 ± 95.0 | <0.001 |
| IFN-γ, pg/mL | 30.2 (undetectable-1416) | 16.8 (undetectable-1245) | 0.003 |
| TGF-β, pg/mL | 939 ± 503 | 1003 ± 463 | 0.243 |
Table 2. Dynamic changes of HBV viral load, HBsAg and cytokine/chemokine levels in CHB patients at baseline and week 12 of PEG-IFN therapy.
In patients with SVR, CXCL9 levels significantly decreased from baseline to week 12, and further decreased at EOT (mean value 228.4, 153.1 and 52.7 pg/mL at baseline, week 12 and EOT, respectively; baseline vs week12, p=0.039; week 12 vs EOT, p=0.208; Figure 1). In patients without SVR, CXCL9 levels remained high at week 12 but decreased later from week 12 to EOT (mean value 126.2, 136.2 and 103.8 pg/mL at baseline, week 12 and EOT, respectively; baseline vs week12, p=0.754; week 12 vs EOT, p=0.005, Figure 1).
In patients with SVR, CXCL9 levels significantly decreased from baseline to week 12, whereas in patients without SVR, CXCL9 levels remained high at week 12 but decreased later from week 12 to EOT. Data were presented as mean ± standard errors of the means (SEM). EOT: end of treatment.
The early CXCL9 levels decline positively correlated with the declines of HBV DNA (r=0.319, p=0.014) and HBsAg levels (r=0.315, p=0.023) at week 12 of PEG-IFN therapy. In contrast, no significant correlations were noted between IP-10 levels decline and declines of viral load or HBsAg levels.
Correlation between serum ALT and chemokine levels
Serum ALT, CXCL9 and IP-10 levels were measured at each time point during PEG-IFN treatment for correlation analysis (overall n=232). As shown in Figure 2, ALT had positive correlation with both serum CXCL9 (r=0.335, p<0.001) and IP-10 levels (r=0.423, p<0.001) by Spearman’s test. Serum CXCL9 and IP-10 levels also had a positive correlation with each other (r=0.353, p<0.001).
(A) A positive correlation between CXCL9 and ALT; (B). A positive correlation between IP-10 and ALT. Concentrations of serum ALT, CXCL9 and IP-10 levels were collected at each time point during PEG-IFN treatment (overall n=232).
Baseline predictors of SVR
Compared with patients not achieving SVR, patients with SVR had significantly lower HBV DNA, HBsAg levels and higher CXCL9 levels (Table 1). In contrast, HBV genotypes, precore or BCP mutations, serum IP-10, IFN-γ, TGF-β levels and the four IL28B SNPs were not associated with SVR. In univariate analysis, age, HBV viral loads, HBsAg and CXCL9 levels were factors associated with SVR (Table 3). In multivariate analysis, HBV DNA <2.5 x 107 IU/mL and CXCL9 >80 pg/mL were pre-treatment predictors of SVR (Table 3).
| Univariate | Multivariate | ||||||
|---|---|---|---|---|---|---|---|
| OR | 95% CI | p | OR | 95% CI | p | ||
| Pretreatment predictor | |||||||
| Age (years) | >40 vs ≤40 | 3.360 | 1.063-10.620 | 0.039 | NS | ||
| Sex | male vs female | 1.161 | 0.327-4.118 | 0.818 | NA | ||
| HBeAg status | positive vs negative | 0.523 | 0.179-1.528 | 0.236 | NA | ||
| Treatment duration | 24 vs 48 weeks | 0.596 | 0.198-1.799 | 0.359 | NA | ||
| HBV genotype | B vs C | 1.238 | 0.407-3.766 | 0.707 | NA | ||
| BCP mutation | mutant vs wild type | 0.734 | 0.229-2.356 | 0.603 | NA | ||
| Precore mutation | mutant vs wild type | 2.385 | 0.814-6.989 | 0.113 | NA | ||
| IL28B polymorphisms | |||||||
| rs8105790 | CC vs CT/TT | - | - | NS* | NA | ||
| rs12979860 | TT vs CT/TT | - | - | NS* | NA | ||
| rs8099917 | GG vs GT/TT | - | - | NS* | NA | ||
| rs10853728 | CC vs CG/GG | 1.149 | 0.355-3.721 | 0.817 | NA | ||
| HBV DNA (IU/mL) | >2.5 x 107 vs ≤2.5 x 107 | 0.062 | 0.008-0.497 | 0.009 | 0.077 | 0.009-0.654 | 0.019 |
| HBsAg (IU/mL) | >2000 vs ≤2000 | 0.240 | 0.073-0.786 | 0.018 | NS | ||
| ALT (U/L) | >400 vs ≤400 | 1.875 | 0.403-8.729 | 0.423 | NA | ||
| CXCL9 (pg/mL) | >80 vs ≤80 | 8.200 | 2.500-26.894 | 0.001 | 6.791 | 1.924-23.967 | 0.003 |
| IP-10 (pg/mL) | >80 vs ≤80 | 1.059 | 0.360-3.116 | 0.917 | NA | ||
| IFN-γ (pg/mL) | >40 vs ≤40 | 0.427 | 0.124-1.469 | 0.177 | NA | ||
| TGF-β (pg/mL) | >1000 vs ≤1000 | 1.887 | 0.657-5.433 | 0.238 | NA | ||
| On-treatment predictor (week 12) | |||||||
| HBV DNA (IU/mL) | >2000 vs ≤2000 | 0.154 | 0.043-0.548 | 0.004 | 0.079 | 0.017-0.377 | 0.001 |
| HBsAg (IU/mL) | >1000 vs ≤1000 | 0.142 | 0.040-0.508 | 0.003 | NS | ||
| CXCL9 (pg/mL) | >30 vs ≤30 | 2.414 | 0.599-9.791 | 0.215 | NA | ||
| IP-10 (pg/mL) | >50 vs ≤50 | 3.889 | 0.977-15.473 | 0.054 | NS | ||
| IFN-γ (pg/mL) | >50 vs ≤50 | 0.587 | 0.140-2.470 | 0.468 | NA | ||
| TGF-β (pg/mL) | >1250 vs ≤1250 | 1.891 | 0.590-6.056 | 0.284 | NA | ||
| HBV DNA decline | >2 Log10 vs ≤2 Log10 | 14.400 | 1.763-117.629 | 0.013 | NS | ||
| HBsAg decline | >10% vs ≤10% | - | - | -* | - | - | -* |
| CXCL9 change | decrease vs increase | 2.591 | 0.715-9.387 | 0.147 | NA | ||
| IP-10 change | decrease vs increase | 1.049 | 0.303-3.630 | 0.940 | NA | ||
| IFN-γ change | decrease vs increase | 1.905 | 0.340-10.667 | 0.464 | NA | ||
| TGF-β change | decrease vs increase | 0.961 | 0.296-3.116 | 0.947 | NA | ||
Table 3. Univariate and multivariate analyses of factors associated with sustained virological response.
In HBeAg-positive patients, age, precore mutation, baseline HBV viral loads and CXCL9 levels correlated with SVR in univariate analysis, while HBV DNA <2.5 x 107 IU/mL was the only predictor of SVR in multivariate analysis (Table S1). In HBeAg-negative patients, HBV DNA, HBsAg and CXCL9 levels correlated with SVR in univariate analysis, while CXCL9 >80 pg/mL was the only pre-treatment predictors of SVR in multivariate analysis (Table S2).
On-treatment predictors of SVR
In univariate analysis, HBV viral load and HBsAg levels, HBV DNA decline >2 Log10 and HBsAg decline >10% at week 12 were on-treatment factors associated with SVR (Table 3). None of the patients who did not have HBsAg decline >10% at week 12 achieved SVR. Although significant dynamic changes of IP-10 and IFN-γ levels from baseline to week 12 were noted, none of these factors were associated with SVR. In multivariate analysis, the on-treatment predictors of SVR were HBV DNA <2000 IU/mL and HBsAg decline >10% at week 12 (Table 3).
In HBeAg-positive patients, only HBV DNA decline >2 Log10 was the on-treatment predictor SVR in uni- and multi-variate analyses (Table S1). In HBeAg-negative patients, HBsAg levels and HBsAg decline >10% correlated with SVR in univariate analysis, while HBsAg decline >10% was the only on-treatment predictor of SVR in multivariate analysis (Table S2).
Baseline CXCL9 levels and on-treatment response
We further analyzed the correlation between baseline CXCL9 levels and on-treatment viral suppression, determined by HBV DNA ever less than 20,000 IU/mL during PEG-IFN treatment. The mean baseline CXCL9 levels in patients with or without achieving HBV DNA <20,000 IU/mL during PEG-IFN treatment were 191.6 vs. 68.5 pg/mL, respectively (p=0.004). Among the 28 patients with baseline CXCL9 >80 pg/mL, 24 (85.7%) had ever achieved HBV DNA <20,000 IU/mL, whereas in the 46 patients with baseline CXCL9 <80 pg/mL, only 26 (56.5) had ever achieved HBV DNA <20,000 IU/mL (p=0.019).
Predictive values of baseline CXCL9 levels on SVR
In order to identify patients who had higher chance of SVR before PEG-IFN treatment, an algorithm based on combination of baseline serum CXCL9 and HBV DNA levels was established (Figure 3). Patients with baseline high viral loads generally had a low chance of SVR. Among 47 patients with low viral loads, 13 of 22 patients with CXCL9 >80 pg/mL achieved SVR (positive predictive value (PPV)=59.1%), whereas only 5 of 25 (20%) patients with CXCL9 <80 pg/mL had SVR (Figure 3A). The SVR rate was lowest (0%) in patients with baseline low CXCL9 levels but high viral loads (negative predictive value (NPV) = 100%). The predictive value of CXCL9 was not dominant in HBeAg-positive patients (Figure 3B), but the performance of CXCL9 in predicting SVR was good in HBeAg-negative patients with low viral loads (PPV=64.3%, Figure 3C).
The prediction algorithm applied for all (A), HBeAg-positive (B) and HBeAg-negative CHB patients (C).
Discussion
Interferon therapy for CHB remains a clinical challenge. Although successful PEG-IFN treatment may induce a durable response and reduce liver-related complications, most of patients could not achieve virological response [10–13], and only a minority of patients choose PEG-IFN as initial antiviral treatment for CHB due to the concern of side effects in Taiwan. Therefore, individualized treatment strategies according to pretreatment and on-treatment parameters should be developed to identify patients with a highest chance of response. However, currently there is no ideal pre-treatment predictor of SVR, especially in HBeAg-negative patients. In this prospective pilot study, we demonstrated the dynamic changes of serum cytokines and chemokines during PEG-IFN therapy and showed that baseline CXCL9 level strongly correlated with SVR in patients with CHB.
In the current study, the HBeAg seroconversion rate and SVR rate in HBeAg-positive patients receiving 24-week PEG-IFN treatment (33.3% and 20%, respectively) were comparable with the results reported in previous studies [11,33,34]. In HBeAg-negative patients, the SVR (31.6%) and HBsAg clearance (5.3%) rates were also consistent with those from the previous reports [35,36].
Declines of serum IP-10 levels have been observed during PEG-IFN therapy in HBeAg-positive CHB patients [37]. In our study, we also observed a significant decline of IP-10 and IFN-γ levels 12 weeks after PEG-IFN treatment, which might reflect a lessened hepatic necro-inflammatory activity after treatment. Although there was no significant change in CXCL9 levels at week 12 in overall patients, patients with SVR had a significant early decline of CXCL9 levels while patients without SVR had a late CXCL9 levels decline (Figure 1). The early CXCL9 decline in patients with SVR might represent an early viral suppression, as supported by the positive correlations between early CXCL9 levels decline and declines of viral load and HBsAg levels. Unfortunately, the early CXCL9 decline could not accurately predict SVR (SVR rate of 35.3% and 17.4% in patients with and without early CXCL9 decline, respectively, p=0.240). Consistent with recent studies showing that HBsAg decline at week 12 strongly correlated with SVR [17,18,38], our data also demonstrated that none of the patients who did not have HBsAg decline >10% at week 12 achieved SVR in this study. HBsAg clearance is a benefit of interferon treatment. It will be interesting to define the relationship between the baseline level of HBsAg and HBsAg clearance, as well as the role of baseline level and dynamic changes of CXCL9 on HBsAg loss. However, during the limited follow-up period of this study, only 2 cases achieved HBsAg clearance. Further large long-term follow-up studies are needed to delineate the role of CXCL9 on HBsAg clearance.
The finding that older patients have higher rates of SVR in univariate analysis might relate to lower viral loads in elder people. Although IL28B SNPs have been shown to predict SVR in patients with chronic hepatitis C and correlate with hepatitis activity in CHB [4,25–27], there was no correlation between IL28B SNPs and SVR in both HBeAg-positive and HBeAg-negative patients. The role of CXCL9 in CHB patients receiving PEG-IFN has not been reported before. CXCL9 and IP-10 were reported to correlate with hepatic injury during hepatic flares in CHB [19]. A strong correlation of serum CXCL9 and IP-10 concentrations with ALT levels was also noted [19]. Consistent with previous report, our results also showed positive correlations between CXCL9, IP-10 and ALT levels. The positive correlations between these parameters may imply that these chemokines have positive impact on the liver inflammation and HBV control, albeit the relevance was not good due to complex interactions between other host and viral factors that contribute the liver inflammation. These findings suggest that both CXCL9 and IP-10 are associated with hepatic necro-inflammation and may be surrogates of the host immune response against HBV infection. IP-10 has been shown to correlate with SVR to IFN-based therapy in patients with HCV infection [20–22]. Sonneveld, et al recently reported that high pretreatment serum IP-10 levels were associated with HBeAg loss after 52 weeks of PEG-IFN therapy in HBeAg-positive patients, albeit the predictive value for combined serological and virological response was less prominent [37]. Different HBV genotypes and duration of PEG-IFN treatment may influence the predictive role of IP-10 in HBeAg-positive patients.
Baseline CXCL9 level significantly correlates not only with SVR, but also with on-treatment viral suppression. This finding supports a stronger host immune response at baseline having a higher chance of SVR under interferon treatment. A recent finding also showed that HBeAg-negative CHB patients with greater baseline HBV-specific CTL responses had a better on-treatment control of HBV replication during PEG-IFN treatment [39].
CXCL9 and IP-10 are chemokines that bind to the cell surface chemokine receptor CXCR3, which is highly expressed on effector T cells and plays an important role in T cell trafficking and function [40,41]. An animal study showed that HBV-specific cytotoxic T lymphocytes (CTLs) induced CXCL9 and IP-10 production and subsequently recruited host inflammatory cells responsible for liver damage [42,43]. Lower levels of CXCL9 and IP-10 correlate with HBV persistence in an animal model [44]. In human study, elevated CXCL9 and IP-10 involve in liver inflammation during hepatitis flares in CHB [19]. Our results showed a positive correlation between CXCL9 and IP-10 levels, representing that both chemokines were induced and participated in immune responses against HBV infection. Although both CXCL9 and IP-10 are ligands of CXCR3, they have distinct properties. IP-10 is induced by a variety of innate stimuli that can further induce IFN-α/β, and is responsible for recruiting type-1 helper (Th1)-type CD4+ T cells, effector CD8+ T cells and innate-type lymphocytes, such as natural killer (NK) and NKT cells. In contrast, the induction of CXCL9 is restricted to IFN-γ, and its function is predominantly in recruiting CD8+ cytotoxic T lymphocytes [41]. The underlying mechanism of CXCL9 level correlates with SVR deserves further study.
The strong correlation of baseline CXCL9 levels with SVR indicates that CXCL9 might serve as a marker to select CHB patients suitable for PEG-IFN therapy, especially in HBeAg-negative cases with low viral loads. In contrast, the SVR rate in patients with high viral loads was low regardless of their baseline CXCL9 levels, suggesting that host antiviral immunity might be overwhelmed by high viral loads.
As a pilot study, the case number was small. It was also due to fewer than 5% of CHB patients in Taiwan had the will to receive PEG-IFN as initial antiviral treatment. Therefore, it is important to identify a pre-treatment marker to predict response to PEG-IFN, not only to encourage patients to receive PEG-IFN treatment but also to save the medical resources for those with a high chance of response. Further large and long-term follow up studies are needed to determine the appropriate cutoff level of CXCL9, and to observe the predictive value of CXCL9 on delayed virological response and HBsAg clearance.
In conclusion, CXCL9 might be used to select appropriate CHB patients to receive PEG-IFN treatment, especially in HBeAg-negative patients with low viral loads.
Supporting Information
Table S1.
Univariate and multivariate analyses of factors associated with sustained virological response in HBeAg-positive patients (n=36).
https://doi.org/10.1371/journal.pone.0076798.s001
(DOC)
Table S2.
Univariate and multivariate analyses of factors associated with sustained virological response in HBeAg-negative patients (n=38).
https://doi.org/10.1371/journal.pone.0076798.s002
(DOC)
Acknowledgments
The authors thank Clinical Research Core Laboratory, Taipei Veterans General Hospital for providing their facilities to help conduct this study.
Author Contributions
Conceived and designed the experiments: YHH. Performed the experiments: ICL. Analyzed the data: ICL. Contributed reagents/materials/analysis tools: YHH CWS YJW TIH KCL HCL. Wrote the manuscript: ICL.
References
- 1. Ganem D, Prince AM (2004) Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med 350: 1118-1129. doi:https://doi.org/10.1056/NEJMra031087. PubMed: 15014185.
- 2. Liaw YF, Chu CM (2009) Hepatitis B virus infection. Lancet 373: 582-592. doi:https://doi.org/10.1016/S0140-6736(09)60207-5. PubMed: 19217993.
- 3. Liaw YF (2009) Natural history of chronic hepatitis B virus infection and long-term outcome under treatment. Liver Int 29 Suppl 1: 100-107. doi:https://doi.org/10.1111/j.1478-3231.2008.01941.x. PubMed: 19207972.
- 4. Lee IC, Lin CH, Huang YH, Huo TI, Su CW et al. (2013) IL28B polymorphism correlates with active hepatitis in patients with HBeAg-negative chronic hepatitis B. PLOS ONE 8: e58071. doi:https://doi.org/10.1371/journal.pone.0058071. PubMed: 23469142.
- 5. Bertoletti A, Ferrari C (2012) Innate and adaptive immune responses in chronic hepatitis B virus infections: towards restoration of immune control of viral infection. Gut 61: 1754-1764. doi:https://doi.org/10.1136/gutjnl-2011-301073. PubMed: 22157327.
- 6. Lok AS, McMahon BJ (2009) Chronic hepatitis B: update 2009. Hepatology 50: 661-662. doi:https://doi.org/10.1002/hep.23190. PubMed: 19714720.
- 7.
European Association For The Study Of The Liver (2012) EASL clinical practice Guidelines: Management of chronic hepatitis B virus infection. J Hepatol 57: 167-185. PubMed: 22436845.
- 8. Sung JJ, Tsoi KK, Wong VW, Li KC, Chan HL (2008) Meta-analysis: Treatment of hepatitis B infection reduces risk of hepatocellular carcinoma. Aliment Pharmacol Ther 28: 1067-1077. doi:https://doi.org/10.1111/j.1365-2036.2008.03816.x. PubMed: 18657133.
- 9. van Zonneveld M, Honkoop P, Hansen BE, Niesters HG, Darwish Murad S et al. (2004) Long-term follow-up of alpha-interferon treatment of patients with chronic hepatitis B. Hepatology 39: 804-810. doi:https://doi.org/10.1002/hep.20128. PubMed: 14999700.
- 10. Perrillo R (2009) Benefits and risks of interferon therapy for hepatitis B. Hepatology 49: S103-S111. doi:https://doi.org/10.1002/hep.22956. PubMed: 19399806.
- 11. Lau GK, Piratvisuth T, Luo KX, Marcellin P, Thongsawat S et al. (2005) Peginterferon Alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B. N Engl J Med 352: 2682-2695. doi:https://doi.org/10.1056/NEJMoa043470. PubMed: 15987917.
- 12. Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S et al. (2004) Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B. N Engl J Med 351: 1206-1217. doi:https://doi.org/10.1056/NEJMoa040431. PubMed: 15371578.
- 13. Janssen HL, van Zonneveld M, Senturk H, Zeuzem S, Akarca US et al. (2005) Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet 365: 123-129. doi:https://doi.org/10.1016/S0140-6736(05)17701-0. PubMed: 15639293.
- 14. Buster EH, Hansen BE, Lau GK, Piratvisuth T, Zeuzem S et al. (2009) Factors that predict response of patients with hepatitis B e antigen-positive chronic hepatitis B to peginterferon-alfa. Gastroenterology 137: 2002-2009. doi:https://doi.org/10.1053/j.gastro.2009.08.061. PubMed: 19737568.
- 15. Wong DK, Cheung AM, O’Rourke K, Naylor CD, Detsky AS et al. (1993) Effect of alpha-interferon treatment in patients with hepatitis B e antigen-positive chronic hepatitis B. A meta-analysis. Ann Intern Med 119: 312-323. doi:https://doi.org/10.7326/0003-4819-119-4-199308150-00011. PubMed: 8328741.
- 16. Fried MW, Piratvisuth T, Lau GK, Marcellin P, Chow WC et al. (2008) HBeAg and hepatitis B virus DNA as outcome predictors during therapy with peginterferon alfa-2a for HBeAg-positive chronic hepatitis B. Hepatology 47: 428-434. doi:https://doi.org/10.1002/hep.22065. PubMed: 18220290.
- 17. Liaw YF (2011) Clinical utility of hepatitis B surface antigen quantitation in patients with chronic hepatitis B: a review. Hepatology 53: 2121-2129. doi:https://doi.org/10.1002/hep.24364. PubMed: 21503943.
- 18. Brunetto MR, Moriconi F, Bonino F, Lau GK, Farci P et al. (2009) Hepatitis B virus surface antigen levels: a guide to sustained response to peginterferon alfa-2a in HBeAg-negative chronic hepatitis B. Hepatology 49: 1141-1150. doi:https://doi.org/10.1002/hep.22760. PubMed: 19338056.
- 19. Tan AT, Koh S, Goh W, Zhe HY, Gehring AJ et al. (2010) A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B. J Hepatol 52: 330-339. doi:https://doi.org/10.1016/S0168-8278(10)60847-1. PubMed: 20137825.
- 20. Lagging M, Romero AI, Westin J, Norkrans G, Dhillon AP et al. (2006) IP-10 predicts viral response and therapeutic outcome in difficult-to-treat patients with HCV genotype 1 infection. Hepatology 44: 1617-1625. doi:https://doi.org/10.1002/hep.21407. PubMed: 17133471.
- 21. Askarieh G, Alsiö A, Pugnale P, Negro F, Ferrari C et al. (2010) Systemic and intrahepatic interferon-gamma-inducible protein 10 kDa predicts the first-phase decline in hepatitis C virus RNA and overall viral response to therapy in chronic hepatitis C. Hepatology 51: 1523-1530. doi:https://doi.org/10.1002/hep.23509. PubMed: 20186843.
- 22. Kurelac I, Lepej SZ, Grlgic I, Gorenec L, Papic N et al. (2012) Chemokine CXCL10 at week 4 of treatment predicts sustained virological response in patients with chronic hepatitis C. J Interferon Cytokine Res Off J International Society For Interferon Cytokine Res 32: 386-391. doi:https://doi.org/10.1089/jir.2012.0006. PubMed: 22799464.
- 23. Chou YC, Chen ML, Hu CP, Chen YL, Chong CL et al. (2007) Transforming growth factor-beta1 suppresses hepatitis B virus replication primarily through transcriptional inhibition of pregenomic RNA. Hepatology 46: 672-681. doi:https://doi.org/10.1002/hep.21726. PubMed: 17580335.
- 24. Hong MH, Chou YC, Wu YC, Tsai KN, Hu CP et al. (2012) Transforming growth factor-beta1 suppresses hepatitis B virus replication by the reduction of hepatocyte nuclear factor-4alpha expression. PLOS ONE 7: e30360. doi:https://doi.org/10.1371/journal.pone.0030360. PubMed: 22276183.
- 25. Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV et al. (2009) Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 461: 399-401. doi:https://doi.org/10.1038/nature08309. PubMed: 19684573.
- 26. Suppiah V, Moldovan M, Ahlenstiel G, Berg T, Weltman M et al. (2009) IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat Genet 41: 1100-1104. doi:https://doi.org/10.1038/ng.447. PubMed: 19749758.
- 27. Tanaka Y, Nishida N, Sugiyama M, Kurosaki M, Matsuura K et al. (2009) Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat Genet 41: 1105-1109. doi:https://doi.org/10.1038/ng.449. PubMed: 19749757.
- 28. Sonneveld MJ, Wong VW, Woltman AM, Wong GL, Cakaloglu Y et al. (2012) Polymorphisms Near IL28B and Serologic Response to Peginterferon in HBeAg-Positive Patients With Chronic Hepatitis B. Gastroenterology 142: 513-520 e511 doi:https://doi.org/10.1053/j.gastro.2011.11.025. PubMed: 22108195.
- 29. Huang YH, Wu JC, Chang TT, Sheen IJ, Lee PC et al. (2003) Analysis of clinical, biochemical and viral factors associated with early relapse after lamivudine treatment for hepatitis B e antigen-negative chronic hepatitis B patients in Taiwan. J Viral Hepat 10: 277-284. doi:https://doi.org/10.1046/j.1365-2893.2003.00428.x. PubMed: 12823594.
- 30. Huang YH, Wu JC, Chang TT, Sheen IJ, Huo TI et al. (2006) Association of core promoter/precore mutations and viral load in e antigen-negative chronic hepatitis B patients. J Viral Hepat 13: 336-342. doi:https://doi.org/10.1111/j.1365-2893.2005.00688.x. PubMed: 16637865.
- 31. Yu ML, Huang CF, Huang JF, Chang NC, Yang JF et al. (2011) Role of interleukin-28B polymorphisms in the treatment of hepatitis C virus genotype 2 infection in Asian patients. Hepatology 53: 7-13. doi:https://doi.org/10.1002/hep.23976. PubMed: 21254157.
- 32. Tillmann HL, Thompson AJ, Patel K, Wiese M, Tenckhoff H et al. (2010) A polymorphism near IL28B is associated with spontaneous clearance of acute hepatitis C virus and jaundice. Gastroenterology 139: 1586-1592, e1581 doi:https://doi.org/10.1053/j.gastro.2010.07.005. PubMed: 20637200.
- 33. Liaw YF, Jia JD, Chan HL, Han KH, Tanwandee T et al. (2011) Shorter durations and lower doses of peginterferon alfa-2a are associated with inferior hepatitis B e antigen seroconversion rates in hepatitis B virus genotypes B or C. Hepatology 54: 1591-1599. doi:https://doi.org/10.1002/hep.24555. PubMed: 22045673.
- 34. Cooksley WG, Piratvisuth T, Lee SD, Mahachai V, Chao YC et al. (2003) Peginterferon alpha-2a (40 kDa): an advance in the treatment of hepatitis B e antigen-positive chronic hepatitis B. J Viral Hepat 10: 298-305. doi:https://doi.org/10.1046/j.1365-2893.2003.00450.x. PubMed: 12823597.
- 35. Marcellin P, Bonino F, Lau GK, Farci P, Yurdaydin C et al. (2009) Sustained response of hepatitis B e antigen-negative patients 3 years after treatment with peginterferon alpha-2a. Gastroenterology 136: 2169-2179 e2161-2164 doi:https://doi.org/10.1053/j.gastro.2009.03.006. PubMed: 19303414.
- 36. Peng CY, Lai HC, Li YF, Su WP, Chuang PH et al. (2012) Early serum HBsAg level as a strong predictor of sustained response to peginterferon alfa-2a in HBeAg-negative chronic hepatitis B. Aliment Pharmacol Ther 35: 458-468. doi:https://doi.org/10.1111/j.1365-2036.2011.04973.x. PubMed: 22225574.
- 37. Sonneveld MJ, Arends P, Boonstra A, Hansen BE, Janssen HL (2013) Serum levels of interferon-gamma-inducible protein 10 and response to peginterferon therapy in HBeAg-positive chronic hepatitis B. J Hepatol 58: 898-903. doi:https://doi.org/10.1016/j.jhep.2013.01.029. PubMed: 23376362.
- 38. Rijckborst V, Hansen BE, Cakaloglu Y, Ferenci P, Tabak F et al. (2010) Early on-treatment prediction of response to peginterferon alfa-2a for HBeAg-negative chronic hepatitis B using HBsAg and HBV DNA levels. Hepatology 52: 454-461. doi:https://doi.org/10.1016/S0168-8278(10)61177-4. PubMed: 20683945.
- 39. Penna A, Laccabue D, Libri I, Giuberti T, Schivazappa S et al. (2012) Peginterferon-alpha does not improve early peripheral blood HBV-specific T-cell responses in HBeAg-negative chronic hepatitis. J Hepatol 56: 1239-1246. doi:https://doi.org/10.1016/j.jhep.2011.12.032. PubMed: 22326467.
- 40. Groom JR, Luster AD (2011) CXCR3 in T cell function. Exp Cell Res 317: 620-631. doi:https://doi.org/10.1016/j.yexcr.2010.12.017. PubMed: 21376175.
- 41. Groom JR, Luster AD (2011) CXCR3 ligands: redundant, collaborative and antagonistic functions. Immunol Cell Biol 89: 207-215. doi:https://doi.org/10.1038/icb.2010.158. PubMed: 21221121.
- 42. Kakimi K, Lane TE, Wieland S, Asensio VC, Campbell IL et al. (2001) Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes. J Exp Med 194: 1755-1766. doi:https://doi.org/10.1084/jem.194.12.1755. PubMed: 11748277.
- 43. Xia YJ, Zeng D, Xia LM, Yu F, Lin HH et al. (2012) Role of monokine induced by interferon-gamma in liver injury induced by hepatitis B virus in mice. J Viral Hepat 19: 509-518. doi:https://doi.org/10.1111/j.1365-2893.2011.01581.x. PubMed: 22676364.
- 44. Chen SH, Wu HL, Kao JH, Hwang LH (2012) Persistent hepatitis B viral replication in a FVB/N mouse model: impact of host and viral factors. PLOS ONE 7: e36984. doi:https://doi.org/10.1371/journal.pone.0036984. PubMed: 22615863.