Serum Hepatitis B Surface Antigen Levels Research Articles

The correlation between interleukin-21 and the recurrence of chronic hepatitis B after entecavir discontinuation

Objective To study the correlation between interleukin (IL)-21 and the recurrence of chronic hepatitis B (CHB) in hepatitis B e antigen (HBeAg)-positive patients after discontinuation of entecavir (ETV). Methods A total of 112 HBeAg-positive CHB patients treated with ETV were enrolled. Serum levels of IL-21 at week 0, 12, 24 and 52 after ETV discontinuation were detected. The Cox regression model was used to analyze the correlation between IL-21 and the recurrence after ETV discontinuation. The receiver operating characteristic (ROC) curve was applied to determine the predictive value of IL-21 for CHB recurrence after ETV discontinuation. The expression differences of IL-21 were compared between sustained viral response (SVR) group and viral relapse (VR) group. The t test and Chi-square test were used for statistical analysis. Results The serum levels of IL-21 in SVR group at week 0, 12, 24 and 52 after ETV withdrawal were (60.0±10.8), (60.2±14.7), (60.6±19.5) and (61.2±15.4) ng/L, respectively, which were all significantly higher than those in VR group (47.5±10.7), (47.3±12.9), (46.9±12.2) and (46.4±12.8) ng/L, respectively (t=6.153, 4.926, 4.382 and 5.515, respectively, all P<0.01). The area under curve (AUC) was 0.811 (95%CI: 0.728~0.893, P<0.01) and the best cut-off value of serum IL-21 level was 49.8 ng/L. The recurrence rates of patients with serum IL-21 level ≥49.8 ng/L and <49.8 ng/L at time of ETV withdrawal was 25.4% (16/63) and 77.6% (38/49), respectively. The difference was statistically significant (χ2=30.027, P<0.01). The serum IL-21 level at the time of drug withdrawal (P=0.005), serum hepatitis B surface antigen (HBsAg) level at the time of HBeAg seroconversion (P=0.008) and age (P=0.016) were factors associated with CHB recurrence after ETV withdrawal by multivariate Cox model analysis. The serum levels of ALT, HBV DNA, HBeAg and HBsAg in SVR group were significantly lower than those of VR group at week 12, 24 and 52 after ETV withdrawal (t=-5.968, -7.691, -8.093; -3.047, -9.477, -28.900; -2.872, -10.424, -18.330; -4.633, -4.030 and -5.032, respectively; all P<0.01). Serum level of IL-21 was negatively correlated with HBsAg in SVR group after ETV withdrawal (r=-0.241, P<0.01), while positively correlated with HBsAg in VR group (r=0.286, P<0.01). Conclusions The serum IL-21 level at the time of drug withdrawal is associated with the recurrence after ETV discontinuation. IL-2l may play an important role as an immunomodulatory factor in maintaining virological responses in HBeAg-positive CHB patients after ETV withdrawal. Key words: Cytokines; Hepatitis B, chronic; Recurrence; Entecavir; Withdrawal

Time-varying serum gradient of hepatitis B surface antigen predicts risk of relapses after off-NA therapy

BackgroundThe serum gradient of hepatitis B surface antigen (HBsAg) varies over time after cessation of nucleos(t)ide analog (NA) treatment in patients with chronic hepatitis B (CHB). The association between the time-varying HBsAg serum gradient and risk of relapse has not been elucidated.MethodsThis multicenter cohort study prospectively enrolled CHB patients who discontinued 3 year-NA treatment. Eligible patients were serologically negative for HBeAg and viral DNA at NA cessation. The participants (n = 140) were followed every 3 months through HBsAg quantification. Virological and clinical relapses were defined as viral DNA levels >2000 IU/mL and alanine aminotransferase (ALT) levels >80 U/mL, respectively. The association of time-varying HBsAg levels with relapses was assessed through a time-dependent Cox analysis.ResultsDuring a median follow-up of 19.9 (interquartile range [IQR], 10.6–25.3) months, virological and clinical relapses occurred in 94 and 49 patients, with a 2-year cumulative incidence of 79.2% (95% confidence interval [CI], 70.9%–86.4%) and 42.9% (95% CI, 34.1%–52.8%), respectively. The serum level of HBsAg was associated with virological (P < 0.001) and clinical (P = 0.01) relapses in a dose–response manner, with adjusted hazard ratios of 2.10 (95% CI, 1.45–3.04) and 2.32 (95% CI, 1.28–4.21). Among the patients (n = 19) whose HBsAg levels ever dropped below 10 IU/mL, only one and three patients subsequently developed clinical and virological relapses.ConclusionThe serum gradient of HBsAg measured throughout the off-therapy observation is associated with the subsequent occurrence of virological and clinical relapses in CHB patients who discontinue NA treatment.

Potential use of serum HBV RNA in antiviral therapy for chronic hepatitis B in the era of nucleos(t)ide analogs.

Although the efficacy of nucleos(t)ide analogue (NA) has been confirmed for treatment of chronic hepatitis B, long-term therapy has been recommended due to the high frequency of off-therapy viral DNA rebound and disease relapse. In this review, the RNA virion-like particles of hepatitis B virus (HBV) are integrated into the life cycle of HBV replication, and the potential significance of serum HBV RNA is systematically described. The production of HBV RNA virion-like particles should not be blocked by NA; in this regard, serum HBV RNA is found to be a suitable surrogate marker for the activity of intrahepatic covalently closed circular DNA (cccDNA), particularly among patients receiving NA therapy. Therefore, the concept of virological response is redefined as persistent loss of serum HBV DNA and HBV RNA. In contrast to hepatitis B surface antigen (HBsAg) that can originate from either the cccDNA or the integrated HBV DNA fragment, serum HBV RNA, with pregenomic RNA origination, can only be transcribed from cccDNA. Therefore, the loss of serum HBV RNA would likely be a promising predicator for safe drug discontinuation. The clinical status of consistent loss of serum HBV RNA accompanied with low serum HBsAg levels might be implicated as a "para-functional cure," a status nearly close to the functional cure of chronic hepatitis B, to distinguish the "functional cure" characterized as serum HBsAg loss with or without anti-HBs seroconversion.

Trajectories of serum hepatitis B surface antigen kinetics in patients with chronic hepatitis B receiving long-term nucleos(t)ide analogue therapy.

The kinetics of serum hepatitis B surface antigen (HBsAg) levels during long-term nucleos(t)ide analogue (NA) therapy in chronic hepatitis B (CHB) patients remains unclear. We investigated the patterns of serum HBsAg kinetics and their association with therapeutic outcomes in genotype B- or C-infected CHB patients receiving long-term NA therapy. We enrolled 329 treatment-naive CHB patients receiving NA therapy for >5years to analyse the kinetic patterns by using group-based trajectory models (GBTMs). Most patients (82.4%) received entecavir therapy. The median treatment duration was 83.6 (68.5-89.7) months. The GBTMs revealed three groups for both the hepatitis B e antigen (HBeAg)-positive and -negative patients. The median annual decline in serum HBsAg levels during the first 5years was significantly higher in Group 1 than in Groups 2 and 3 in HBeAg-positive (0.78 vs 0.10 vs 0.10 log10 IU/mL) and HBeAg-negative (0.71 vs 0.08 vs 0.09 log10 IU/mL) patients. HBsAg levels at the baseline and 12months combined with an HBsAg decline from the baseline to 12months of treatment predicted trajectory pattern 1 in HBeAg-positive (sensitivity, 77.8%; specificity, 99.1%; positive predictive value [PPV], 87.5%; and negative predictive value [NPV], 98.2%) and HBeAg-negative (sensitivity, 100%; specificity, 99.5%; PPV, 88.9%; and NPV, 100%) patients. The trajectory patterns were significantly associated with HBeAg loss in the HBeAg-positive patients and the achievement of HBsAg <100IU/mL or HBsAg loss in HBeAg-positive and HBeAg-negative patients. The trajectory of serum HBsAg levels predicts HBsAg loss in CHB patients receiving long-term NA therapy.

Smaller reduction of hepatitis B virus DNA in liver tissue than in serum in patients losing HBeAg.

The prognosis and outcome of treatment for chronic hepatitis B virus (HBV) infection are predicted by levels of HBV DNA in serum. These levels are composed of relaxed circular DNA (rcDNA) and double stranded linear DNA in viral particles, whereas, HBV DNA in liver tissue also can be covalently closed circular DNA (cccDNA) or integrated into the human genome. The aim of this study was to investigate the quantitative relation between HBV DNA in serum and tissue, its change over time and how these markers relate to serum levels of hepatitis B surface antigen (HBsAg). Serum and liver biopsies taken from 15 patients with chronic HBV infection on two occasions during 2.7-11.1 years were analyzed retrospectively. At baseline, the median HBV DNA levels in serum were 7.76 log10 IU/mL in nine hepatitis B e antigen (HBeAg) positive and 3.65 log10 IU/mL in six HBeAg-negative patients. At follow-up, serum HBV DNA, serum HBsAg, and intrahepatic HBV DNA (ihDNA) levels had declined by 4.36, 0.52, and 1.47 log10 units, respectively, in seven patients that lost HBeAg, whereas the corresponding reductions were 0.36, 0.30, and 0.39 log10 units in eight patients with unchanged HBeAg status. We conclude that HBV DNA in liver tissue declined almost 1000 times less than HBV DNA in serum during and after loss of HBeAg. This finding raises the possibility that integrated sequences constitute a significant part of the ihDNA. Alternatively, the greater decline of HBV DNA in serum might be due to yet unknown mechanisms acting downstream of reverse transcription.

Association between mitochondrial DNA content and baseline serum levels of HBsAg in chronic hepatitis B infection.

Recent studies have demonstrated a potential link between mitochondrial DNA (mtDNA) content and cirrhosis or hepatocellular carcinoma (HCC). However, there are few studies evaluating mtDNA content as a noninvasive marker of chronic hepatitis B infection (CHB). In this study, we conducted a case-control study to determine mtDNA content in peripheral blood leukocyte (PBL) samples from 76 CHB cases naïve to antivirus therapy and 96 healthy controls, and then evaluated the association between mtDNA content and baseline serum concentration of HBV markers. Consequently, CHB cases had significantly higher mtDNA content than healthy controls (1052.85 vs 618.98, P < 0.001). Pearson's correlation analysis revealed that mtDNA content was negatively correlated with the baseline levels of hepatitis B surface antigen (HBsAg) (r = -0.291, P = 0.011) in CHB patients. In a trend analysis, a statistically significant association was detected between lower mtDNA content and increasing levels of HBsAg (P = 0.015). In conclusion, our study provides the first epidemiological evidence that mtDNA content of CHB cases naive to antivirus therapy is significantly higher than healthy controls and the levels of mtDNA content is negatively associated with HBsAg. mtDNA content may serve as a potential noninvasive biomarker of CHB which may need more researches to validate.

Serum Hepatitis B Virus DNA, RNA, and HBsAg: Which Correlated Better with Intrahepatic Covalently Closed Circular DNA before and after Nucleos(t)ide Analogue Treatment?

The study was designed to investigate whether serum hepatitis B virus (HBV) RNA is a strong surrogate marker for intrahepatic HBV covalently closed circular DNA (cccDNA) compared with serum HBV DNA, hepatitis B surface antigen (HBsAg), and hepatitis B e antigen (HBeAg) in HBeAg-positive chronic hepatitis B (CHB) patients. Serum HBV RNA, HBV DNA, HBsAg, HBeAg, and intrahepatic cccDNA were quantitatively detected at baseline (n = 82) and 96 weeks (n = 62) after treatment with nucleos(t)ide analogue (NUC) in HBeAg-positive CHB patients. The correlations among serum HBV RNA, HBV DNA, HBsAg, HBeAg, and intrahepatic cccDNA levels were then statistically analyzed. The results showed that pretreatment intrahepatic cccDNA levels correlated better with serum HBV DNA levels (r = 0.36, P < 0.01) than with serum HBV RNA levels (r = 0.25, P = 0.02), whereas no correlations were found between pretreatment intrahepatic cccDNA levels and HBsAg (r = 0.15, P = 0.17) or HBeAg (r = 0.07, P = 0.56) levels. At 96 weeks after NUC treatment, intrahepatic cccDNA levels correlated well with HBsAg levels (r = 0.39, P < 0.01) but not with serum HBV RNA, HBV DNA, and HBeAg levels (all P > 0.05). Besides, the decline in the intrahepatic cccDNA level from baseline to week 96 correlated better with the reduction in the serum HBsAg levels than with the decreases in the levels of the other markers (for the HBsAg decline, r = 0.38, P < 0.01; for the HBV DNA decline, r = 0.35, P = 0.01; for the HBV RNA decline, r = 0.28, P < 0.05; for the HBeAg decline, r = 0.18, P = 0.19). In conclusion, the baseline serum HBV RNA level or its decline after 96 weeks of NUC therapy correlated with the corresponding intrahepatic cccDNA level, while it was less than that seen with serum HBV DNA at baseline and HBsAg (or its decline) at 96 weeks after treatment, respectively.

The relationship between serum hepatitis B surface antigen levels and liver pathology during the natural history of chronic hepatitis B

Objective To investigate the relationship between hepatitis B surface antigen (HBsAg) levels and liver pathology at different phases of natural history in chronic hepatitis B (CHB) patients, and to establish a non-invasive liver fibrosis diagnostic model based on HBsAg quantification. Methods A total of 145 CHB patients were enrolled and underwent liver biopsy from January 2013 to January 2015, among which 73 patients were hepatitis B e antigen (HBeAg) positive. HBsAg levels and HBV DNA levels were compared between patients at different phases of natural history and between patients with different HBeAg statuses. Logistic analysis was used to analyze the risk factors associated with fibrosis in HBeAg-positive patients, and to evaluate the predictive value of non-invasive liver fibrosis diagnostic model based on HBsAg quantification. Analysis of variance was used for statistical analysis, and t test analysis was used for the comparison between two independent samples. Results The serum HBsAg levels at the immunologic tolerance phase, immunologic clearance phase, low copy phase and reactivation phase of CHB patients were (4.29±0.69), (3.56±0.61), (3.22±0.64), and (3.54±0.50) lg IU/mL, respectively (F=16.72, P<0.01), and the HBV DNA levels were (8.48±0.58), (6.69±1.44), (3.80±0.59), and (6.21±1.06) lg IU/mL, respectively (F=76.73, P<0.01). In HBeAg-positive CHB patients with liver inflammation stage (G)≤G1, G2, G3 and G4, the serum HBsAg levels were (4.44±0.65), (4.00±0.72), (3.74±0.62), and (3.28±0.50) lg IU/mL, respectively (F=9.198, P<0.01). In HBeAg-positive CHB patients with liver fibrosis stage (S)≤S1, S2, S3, and S4, the serum HBsAg levels were (4.55±0.54), (4.04±0.89), (3.59±0.63), and (3.34±0.50) lg IU/mL, respectively (F=10.66, P<0.01). Logistic analysis showed that age (OR=1.091, 95%CI: 1.013—1.175) and HBsAg level (OR=0.190, 95%CI: 0.066—0.542) were independent factors for predicting fibrosis stage. The area under receiver operating characteristic curve of the non-invasive fibrosis model based on age and HBsAg level was 0.849, which was higher than aspartate aminotransferase to platelet ratio index (0.749) and fibrosis index based on the 4 factors (0.763). Conclusions The serum HBsAg levels are significantly different among the different phases of natural history in CHB patients. The serum HBsAg levels decline with the progression of liver fibrosis in HBeAg-positive CHB patients. The non-invasive diagnostic model that based on HBsAg quantification could be used to evaluate the stage of liver fibrosis. Key words: Hepatitis B, chronic; Hepatitis B surface antigens; Natural history; Pathological damage

Risk Factors for Prognosis of Hepatocellular Carcinoma After Curative Resection In Patients with Low Hepatitis B Viral Load

BackgroundA retrospective cohort study was conducted to investigate the effect of hepatitis B surface antigen (HBsAg) level on prognosis in low viral load (< 2000 IU/mL) patients with hepatitis B-related hepatocellular carcinoma (HCC) after curative resection. Material and MethodsA total of 192 patients with low viral load who had received curative resection of pathologically confirmed HCC were analyzed to determine the factors affecting prognosis. The risk factors for survival, early and late recurrence (2 years as a cut-off) were studied. ResultsThe median follow-up time was 38.5 months. The overall survival rates at 1-, 3-, and 5-year after curative resection were 94.2%, 64.0%, and 45.2%, respectively. The cumulative recurrence rates at 1-, 3-, and 5-year after curative resection were 22.4%, 46.5%, and 67.0%, respectively. Patients with high serum HBsAg levels (> 250 IU/mL) had significantly lower survival rates than those with low HBsAg levels (HR: 1.517, 95% CI: 1.005-2.292, P = 0.047). Stratified analysis showed that patients with high HBsAg levels had a significantly higher late recurrence incidence than those with low HBsAg levels (HR: 2.155, 95% CI: 1.094-4.248, P = 0.026), but did not have a significantly higher risk of early recurrence postoperatively (HR: 1.320, 95% CI: 0.837-2.082, P = 0.233). Multivariate analysis revealed that HBsAg > 250 IU/mL was an independent risk factor associated with late recurrence (HR: 2.109, 95% CI: 1.068-4.165, P = 0.032). ConclusionsHBsAg > 250 IU/mL at the time of tumor resection was an independent risk factor for late recurrence in low viral load HCC patients.

Different Mechanisms May Exist for HBsAg Synthesis and Secretion During Various Phases of Chronic Hepatitis B Virus Infection.

BackgroundThe aim of this study was to characterize the expression and secretion of hepatitis B surface-antigen (HBsAg) in the hepatocytes of hepatitis B virus (HBV)-infected patients at different phases of infection; as such, the association of intrahepatic HBsAg expression with virological markers and the histological characteristics were analyzed.Material/Methods302 chronic HBV infection patients who had not received antiviral therapy were stratified by HBeAg status. The proportion of HBsAg-positive cells was used as an indicator for HBsAg expression level.ResultsIn HBeAg-positive patients, there was a significant correlation between serum HBsAg and serum HBV DNA levels (r=0.569, p<0.001). Intrahepatic HBsAg expression and serum HBsAg level in HBeAg-positive patients were higher than those in HBeAg-negative patients (p=0.002 and p<0.001, respectively). A significant correlation between serum HBsAg level and intrahepatic HBsAg expression was found in HBeAg-negative patients (r=0.377, p<0.001), but not in HBeAg-positive patients (r=0.051, p=0.557). Very interestingly, the correlation between serum HBsAg level and HBsAg expression in hepatocytes gradually increased along with disease progression through the immune-tolerant, immune-clearance, inactive, and recovery phases of HBV infection (r=−0.184, 0.068, 0.492, and 0.575; and p=0,238, 0,722, 0.012, and 0.002, respectively).ConclusionsDifferent mechanisms may be involved in HBsAg synthesis and secretion in different phases of chronic HBV infection.

Expression of microRNA let-7a positively correlates with hepatitis B virus replication in hepatocellular carcinoma tissues.

Let-7a miRNA is downregulated in various cancers. However, in hepatocellular carcinoma (HCC) patients infected with hepatitis B virus (HBV), the relationship between let-7a and HBV replication has not been fully elucidated. Liver specimens were collected from 23 HCC patients with chronically active HBV. The serum levels of the HBV antigens hepatitis B surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg), and the HBV antibodies, anti-HBs, anti-HBe and anti-hepatitis B core antigen (anti-HBc) were measured using the microparticle enzyme immunoassay. Let-7a levels and HBV DNA copy numbers were measured by quantitative real-time PCR (qRT-PCR) and analyzed statistically. A let-7a specific antisense oligonucleotide was introduced to the HBV-producing cell line HepG2.2.15 and a change in HBV DNA copy numbers was assessed by qRT-PCR. HCC patients with highly active HBV replication (>106 DNA copies/mL) showed higher levels of serum HBsAg and anti-HBc than patients with less active HBV replication (<103 DNA copies/mL). The level of let-7a was lower in malignant tissues than in adjacent normal tissues. However, patients with highly active HBV replication demonstrated a significantly higher level of let-7a in hepatocarcinoma tissue than patients with less active HBV replication ( P < 0.05). A higher level of let-7a was observed in the HBV-producing cell line HepG2.2.15 than in HepG2 cells ( P < 0.05), and let-7a down-regulation by antisense oligonucleotides led to a reduction in HBV DNA copy numbers ( P < 0.05), indicating a correlation between the let-7a level and HBV replication. Down-regulation of let-7a reduces HBV replication and could prevent the development of HCC, suggesting it could be an effective therapeutic treatment for HBV infection. Impact statement Although interferon and nucleic acid analogues effectively suppress HBV replication in HBV patients, there is no treatment which eradicates the virus. Moreover, the therapeutic effect can be reduced by virus mutations or drug resistance. Let-7a is a miRNA initially found in the nematode as a master regulator of developmental processes, but also exists in humans. It has been reported that the transcription of let-7a was much lower in HCC than in normal liver tissues and specific miRNA could directly promote virus replication. Therefore we hypothesized that transcription of let-7a promotes HBV replication, which might compromise the therapeutic effects of antivirus treatments. In our present study, we demonstrated a correlation between let-7a transcription and HBV replication in surgical specimens obtained from patients with HCC, as well as in HCC cell lines. Our finding might be the base for a new approach to improve HBV infection treatments in the future.

Expression of Hepatocyte Hepatitis B Core Antigen and Hepatitis B Surface Antigen as a Marker in the Management of Chronic Hepatitis B Patients.

Background/AimsWe aimed to clarify the association of hepatitis B surface antigen (HBsAg)/hepatitis B core antigen (HBcAg) with the disease status and treatment response in patients with chronic hepatitis B (CHB).MethodsWe investigated 171 biopsy-proven entecavir-treated CHB patients (109 hepatitis B e antigen [HBeAg]-positive, 62 HBeAg-negative). HBcAg expression was positive when ≥10% of hepatocytes stained, and classified into nuclear, mixed, and cytoplasmic patterns. HBsAg expressions were intracytoplasmic (diffuse, globular, and submembranous) and membranous. The histologic activity index (HAI) and fibrosis stage followed Ishak system.ResultsIn HBeAg-positive patients, older age, increased HAI score, advanced fibrosis, and reduced viral load were observed when HBcAg expression shifted from nucleus to cytoplasm in HBcAg-positive patients, and HBsAg expression from non-submembranous to submembranous in HBcAg-negative patients (all, p<0.05). In HBeAg-negative patients, only intracytoplasmic HBsAg expression patterns had clinical relevance with decreased ALT levels and viremia. In HBeAg-positive patients without favorable predictors of virologic response, negative HBcAg and membranous HB-sAg expression predicted greater virologic response (both, p<0.05). The probability of HBeAg seroclearance was higher in patients with increased HAI or lacking HBcAg expression (both, p<0.05). Higher serum HBsAg levels and hepatocyte HBcAg positivity were associated with reduced serum HBsAg during first and post-first year treatment, respectively (both, p<0.05).ConclusionsHepatocyte HBcAg/HBsAg expression is a good marker for disease status and predicting treatment response.

Higher Serum Levels of Type I Interferon Receptor in Adults with Chronic Hepatitis B Leading to Hepatitis B Surface Antigen Clearance

Background: Loss of hepatitis B surface antigen (HBsAg) in chronic hepatitis B (CHB) infection is a favorable outcome. Type I interferon (IFNI) has an essential role to fight virus infections when they bind to the IFN-α/β receptor (IFNAR). Free-circulating IFNARs, known as IFNAR2, perform as carrier proteins to keep the ligands from proteolysis as well as antagonists for ligand binding. Objectives: In this study, we evaluated the HBsAg titer and IFNAR2 in serum baseline of a subcohort of Iranian HBeAg-negative patients with chronic hepatitis B. Methods: Sixty-four patients who spontaneously cleared HBsAg and 100 chronic hepatitis B patients enrolled in this study for assessment of the serum levels of HBsAg and IFNAR2. Results: Serum levels of HBsAg and IFNAR2 were both powerfully associated with loss of HBsAg. The baseline HBsAg titer was significantly lower (333.72 ± 1300 IU/mL vs 3811 ± 6779 IU/mL, P = 0.00) and the IFNAR2 serum level was significantly higher (1.64 ± 0.6 vs 0.87 ± 0.5 ng/mL, P = 0.00) in those who cleared HBsAg compared to the CHB patients. Conclusions: These findings indicated the association of the HBsAg titer and serum IFNAR2 in HBsAg clearance in hepatitis B virus-infected patients. In consequence, immune mechanisms related to IFN-α/β signaling might be responsible in CHB outcome. © 2017, Ahvaz Jundishapur University of Medical Sciences.

Telbivudine versus entecavir in patients with undetectable hepatitis B virus DNA: a randomized trial

BackgroundTelbivudine has been suggested to induce hepatitis B surface antigen (HBsAg) decline to the similar degree as pegylated interferon. We aimed to investigate whether telbivudine could further decrease HBsAg titer in patients who maintain undetectable serum hepatitis B virus (HBV) DNA after initial entecavir treatment.MethodsIn this open-label trial, patients who had serum HBsAg and HBV DNA levels ≥1,000 IU/mL and <60 IU/mL, respectively, following entecavir (0.5 mg/day) treatment for HBeAg-positive chronic hepatitis B were randomized to either switch treatment to telbivudine (600 mg/day, n = 47) or continue entecavir (n = 50) for 48 weeks.ResultsThe baseline characteristics were comparable between groups including HBsAg levels (median, 3.41 log10 IU/mL). All patients had undetectable HBV DNA and normal alanine aminotransferase level. At week 48, the mean change in serum HBsAg levels was not significantly different between the telbivudine and entecavir groups (−0.03 log10 IU/mL vs. −0.05 log10 IU/mL; P = 0.57). No patient experienced HBsAg seroclearance or HBsAg decline >0.5 log10 IU/mL. Eleven patients (23.4%) in the telbivudine group, but none in the entecavir group, experienced virologic breakthrough (P < 0.001). Seven patients (14.9%) exhibited genotypic resistance mutations (M204I +/− L180M) during the virologic breakthrough.ConclusionSequential therapy with entecavir followed by telbivudine resulted in a high rate of virologic breakthrough and drug-resistance without any beneficial effect on HBsAg decline. These results do not support the use of low genetic barrier drugs as a switch treatment strategy in patients who achieve virologic response with high genetic barrier drugs.Trial registrationNCT01595685 (date of trial registration: May 8, 2012)

Issue Highlights

Issue Highlights

Hepatitis B surface antigen titer is a good indicator of durable viral response after entecavir off-treatment for chronic hepatitis B

Background/AimsClear indicators for stopping antiviral therapy in chronic hepatitis B (CHB) patients are not yet available. Since the level of hepatitis B surface antigen (HBsAg) is correlated with covalently closed circular DNA, the HBsAg titer might be a good indicator of the off-treatment response. This study aimed to determine the relationship between the HBsAg titer and the entecavir (ETV) off-treatment response.MethodsThis study analyzed 44 consecutive CHB patients (age, 44.6±11.4 years, mean±SD; men, 63.6%; positive hepatitis B envelope antigen (HBeAg) at baseline, 56.8%; HBV DNA level, 6.8±1.3 log10 IU/mL) treated with ETV for a sufficient duration and in whom treatment was discontinued after HBsAg levels were measured. A virological relapse was defined as an increase in serum HBV DNA level of >2000 IU/mL, and a clinical relapse was defined as a virological relapse with a biochemical flare, defined as an increase in the serum alanine aminotransferase level of >2 × upper limit of normal.ResultsAfter stopping ETV, virological relapse and clinical relapse were observed in 32 and 24 patients, respectively, during 20.8±19.9 months of follow-up. The cumulative incidence rates of virological relapse were 36.2% and 66.2%, respectively, at 6 and 12 months, and those of clinical relapse were 14.3% and 42.3%. The off-treatment HBsAg level was an independent factor associated with clinical relapse (hazard ratio, 2.251; 95% confidence interval, 1.076–4.706; P=0.031). When patients were grouped according to off-treatment HBsAg levels, clinical relapse did not occur in patients with an off-treatment HBsAg level of ≤2 log10 IU/mL (n=5), while the incidence rates of clinical relapse at 12 months after off-treatment were 28.4% and 55.7% in patients with off-treatment HBsAg levels of >2 and ≤3 log10 IU/mL (n=11) and >3 log10 IU/mL (n=28), respectively.ConclusionThe off-treatment HBsAg level is closely related to clinical relapse after treatment cessation. A serum HBsAg level of <2 log10 IU/mL is an excellent predictor of a sustained off-treatment response in CHB patients who have received ETV for a sufficient duration.

Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA

As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg. The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements. The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change the ability of HBsAg to bind to antibodies, or impact HBV replication. These could all result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels. Clinical trial number: NCT01088009.

Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), a mini-chromosome essential for HBV replication, is supposed to be resistant to nucleos(t)ide analogue treatment. We investigated the effect of long-term nucleos(t)ide analogue treatment on cccDNA. Among 129 patients who had been enrolled in previous international nucleos(t)ide analogue clinical trials and had liver biopsies at baseline and one year after treatment, we recruited 43 patients on long-term continuous treatment for 72 to 145months for a third liver biopsy. Serum HBV DNA, hepatitis B surface antigen (HBsAg) levels, total intrahepatic HBV DNA (ihHBV DNA), cccDNA, HBV pregenomic RNA (pgRNA) as well as histologic changes were examined. At the time of the third biopsy, serum HBV DNA levels were undetectable in all but one patient. The median levels of HBsAg, ihHBV DNA, and cccDNA were 2.88logIU/ml, 0.03copies/cell, and 0.01copies/cell, respectively. Compared to baseline levels, there was reduction of HBsAg levels by 0.54log (71.46%), ihHBV DNA levels by 2.81log (99.84%), and cccDNA levels by 2.94log (99.89%), with 49% having cccDNA levels below the detection limit. One patient had undetectable HBsAg. The median pgRNA level, measured only in the third biopsy, was 0.021copies/cell, with 40% of patients having undetectable pgRNA. Long-term nucleos(t)ide analogue treatment induced marked depletion of cccDNA in the majority of patients while serum HBsAg levels, though reduced, were detectable in all but one patient. Whether cccDNA depletion is sustained and associated with better patient outcome requires further study. It is generally presumed that a form of hepatitis B virus DNA, called covalently closed circular DNA (cccDNA), which hides inside the nuclei of liver cells of patients with chronic hepatitis B, cannot be reduced by antiviral treatment. The present study showed that with prolonged treatment (median period 126months), cccDNA can be markedly reduced, with 49% of liver biopsies having undetectable cccDNA. This suggests that viral replication capacity would be very low after prolonged antiviral treatment.

Dynamic Characteristics of Serum Hepatitis B Surface Antigen in Chinese Chronic Hepatitis B Patients Receiving 7 Years of Entecavir Therapy.

Background:The ultimate goal of hepatitis B treatment is hepatitis B surface antigen (HBsAg) seroclearance. Several factors have been suggested to be associated with the rate of HBsAg reduction in antiviral-naive or lamivudine therapy cohorts. However, there are few studies evaluating the factors during long-term entecavir (ETV) therapy. In the present study, we aimed to evaluate the factors to predict the outcome of ETV therapy for 7 years.Methods:A total of 47 chronic hepatitis B (CHB) patients treated with ETV monotherapy were included in this study. Liver biochemistry, hepatitis B virus (HBV) serological markers, serum HBV DNA, and HBsAg titers were tested at baseline, 3 months, 6 months, and yearly from 1 to 7. The associations between factors and HBsAg reduction were assessed using multivariate tests with repeated measure analysis of variance.Results:At baseline, serum HBsAg levels showed a positive correlation with baseline HBV DNA levels (r = 0.625, P < 0.001). The mean HBsAg titers after ETV treatment were significantly lower than the baseline titers (P ranges from 0.025 to 0.000,000,6). The HBsAg reduction rate during the 1st year was greater compared to after 1 year of treatment (P < 0.05). Multivariate test showed that hepatitis B e antigen (HBeAg) seroclearance and/or HBsAg reduction ≥0.5 log10 IU/ml at 6 months had a high negative predictive value (96.77%) for HBsAg seroclearance (P = 0.002, P = 0.012, respectively).Conclusions:The HBsAg reduction rate during the 1st year was greater than that after 1 year of treatment. Further, HBeAg status and HBsAg levels at month 6 are the optimal factors for the early prediction of HBsAg seroclearance after long-term ETV therapy in CHB patients.

Association Between Serum Level of Hepatitis B Surface Antigenat End of Entecavir Therapy and Risk of Relapse inEAntigen-Negative Patients.

This study investigated whether serum level of hepatitis B surface antigen (HBsAg) at the end of entecavir treatment was associated with risk of relapse. We performed a prospective multicenter study of 161 consecutive patients with chronic hepatitis B in whom the hepatitis B virus was no longer detected after 3 years or more of entecavir therapy. Treatment ended between July 1, 2011 and July 1, 2015. Patients were monitored for clinical relapse (hepatitis B virus DNA >2000 IU/mL and level of alanine aminotransferase more than 2-fold the upper limit of normal) and virologic relapse (hepatitis B virus DNA >2000 IU/mL). Outcomes were calculated using the Kaplan-Meier method and risk factors were identified by Cox proportional hazards modeling. Two years after therapy ended, 49.2% of patients in the entire cohort had a clinical relapse (95% confidence interval [CI], 40.9%-58.1%) and 81.7% had a virologic relapse (95% CI, 74.3%-88.0%). Among patients who were hepatitis B e antigen-negative at the end of therapy, 39.2% had a clinical relapse (95% CI, 30.3%-49.6%) and 77.4% had a virologic relapse (95% CI, 68.6%-85.2%). Serum level of HBsAg was associated with relapse in the hepatitis B e antigen-negative patients (Ptrend= .006 for clinical relapse; Ptrend= .0001 for virologic relapse). In multivariate Cox regression analysis, the hazard ratio (per log IU/mL increment) forclinical relapse was 2.47 (95% CI, 1.45-4.23) and for virologic relapse was 1.80 (95% CI, 1.33-2.45). The 11 (9%) patients with levels of HBsAg <10 IU/mL did not relapse. Serum level of HBsAg is associated with risk of relapse in patients who are hepatitis B e antigen-negative after treatment with entecavir. A low titer of HBsAg might be used to identify patients at low risk for relapse after treatment.