Serum HBsAg Levels Research Articles

MiR-548c-3p targets TRIM22 to attenuate the Peg–IFN–α therapeutic efficacy in HBeAg-positive patients with chronic hepatitis B

Chronic hepatitis B (CHB) patients treated with interferon shows encouraging results. However, its clinical efficacy is limited by significant individual differences in treatment responses. We identified an interferon-inducible effector, TRIM22, as the likely causal target of such differential responses. We found that TRIM22 was highly expressed in interferon-responsive patients and negatively correlated with HBV DNA and HBeAg serum levels. Stable cells overexpressing TRIM22 carried significantly less HBsAg, HBeAg, and HBV DNA, and cells with knocked-down TRIM22 by shRNA displayed higher levels of these markers than controls. Integrated bioinformatics analysis and subsequent experiments revealed that TRIM22 overexpression significantly increased the supernatant levels of IL-1β and IL-8, two important cytokines of NOD2/NF-κB pathway involved in interferon-induced antiviral activities. We identified three candidate microRNAs binding to 3′UTR of TRIM22 at various locations through typical imperfect paring using the TargetScan program. MiR-548c-3p appeared to be highly expressed, while the TRIM22 level was low in the suboptimal response group of CHB patients. The Luciferase reporter assay revealed an interaction between miR-548c-3p and the 3′UTR of TRIM22, leading to a controlled suppression of TRIM22 endogenous expression. This resulted in interferon's substantially weakened therapeutic efficacy, as indicated by the elevation of the serum levels of HBsAg, HBeAg and HBV DNA in miR-548c-3p-transfected HepAD38 cells. Our study demonstrated that a particular miR-548c-3p is the key negative regulator of TRIM22 in CHB patients with a weak response to interferon treatment, providing a novel marker and target in interferon-α therapy evaluation.

Serum HBsAg and HBcrAg is associated with inflammation in HBeAg-positive chronic hepatitis B patients.

Liver inflammation is the main risk factor for developing liver fibrosis, cirrhosis, and even hepatocellular carcinoma in chronic hepatitis B (CHB) patients. To replace biopsy, additional non-invasive biomarkers to diagnose and grade liver necroinflammation are urgently required in clinical practice. Ninety-four CHB patients, including 74 HBeAg-positive and 20 HBeAg-negative patients, were enrolled and started entecavir or adefovir therapy. Serum HBV RNA, HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), ALT and AST levels, as well as intrahepatic HBV DNA and cccDNA were measured at baseline and during treatment. Liver inflammation was assessed at baseline and month 60 by liver biopsy. Inflammation regression was defined as a ≥1-grade decrease according to the Scheuer scoring system. In HBeAg-positive CHB patients, at baseline, serum HBsAg and HBcrAg levels negatively correlated with inflammation grade, while ALT and AST levels positively correlated with inflammation grade. AST plus HBsAg exhibited excellent diagnostic ability for significant inflammation with an AUROC of 0.896. After 60 months of antiviral treatment, almost all the patients' liver inflammation ameliorated to G1, and no patients had inflammation progression. Besides ALT and AST, serum HBsAg and HBcrAg correlated with inflammation grade in HBeAg-positive CHB patients before NAs treatment. Moreover, the combination of HBsAg and AST exhibited excellent diagnostic ability for significant inflammation.

Antisense oligodeoxynucleotides: the certain but limited efficacy and the uncovering mechanisms for the cure of chronic hepatitis B

Recently, several phase I and phase II clinical trials of antisense oligodeoxynucleotides (ASOs) targeting to the commonly shared conserved sequences of HBV transcripts brought us some promising results. Particularly in the report of phase IIb clinical trial of Bepirovirsen (GSK3228836), approximately 9-10% patients with low baseline serum HBsAg (> 100 IU/ml & < 3 000 IU/ml) achieved functional cure after 24 weeks' of Bepirovirsen treatment. After reviewing the results of other clinical trials, one would be impressed to know that ALG-020572 (Aligos), RO7062931 (Roche) and GSK3389404 (GSK) all failed to sufficiently suppress serum HBsAg expression though the hepatocyte-targeted delivery of these ASOs were enhanced via N-acetyl galactosamine conjugation. Bepirovirsen enabled some patients to achieve sustained disappearance of serum HBsAg. The analysis of its distribution in different tissues of patients after drug administration showed that only a few fractions of ASOs entered liver tissues and far fewer eventually entered hepatocytes. Taking into consideration that only a few hepatocytes could be expected positive for HBsAg staining among these participants with low serum HBsAg level. We suspect that the mechanistic contribution of ASOs declining the serum HBsAg is not only via directly acting on the HBV transcripts in hepatocytes, but also via entering non-parenchymal cells such as Kupffer cells and resulting in stimulation and activation of innate immunity. Eventually the serum HBsAg declines in most participants and even disappears in a small fraction of patients with low baseline HBsAg level, via attack the infected hepatocytes evidenced by the aberrant elevation of ALT. Nevertheless, the functional cure of CHB remains a challenging issue and more efforts are needed.

Magnetic‐Activated Nanosystem with Liver‐Specific CRISPR Nonviral Vector to Achieve Spatiotemporal Liver Genome Editing as Hepatitis B Therapeutics

AbstractChronic hepatitis B infection remains incurable due to the stable presence of various forms of hepatitis B virus (HBV) genome, especially the HBV covalently closed circular DNA (cccDNA). The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) technology provides a new opportunity to potentially cure the HBV infection. However, the efficiency and specificity remain unsatisfactory, especially for nonviral CRISPR/Cas9 delivery. To tackle these, a liver‐specific CRISPR/Cas9 magnetic nanosystem FMNPpAG333/sgXPP is constructed based on fluorinated polyethylenimine‐coated magnetic nanoparticles and liver‐specific ApoE.HCR.hAAT promoter‐driven Cas9‐T2A‐EGFP plasmid with dual sgRNAs. The elaborate system enables magnetic field‐induced spatially specific distribution and hepatocyte‐specific promoter‐driven liver‐specific gene editing. Moreover, this CRISPR/Cas9 magnetic nanosystem is designed to disrupt the two conserved sites in X opening reading frame and Pol opening reading frame of the HBV genome, thereby significantly inactivating the HBV genome without showing off‐target effects. Treatment with FMNPpAG333/sgXPP for 7 days reduces serum HBsAg levels by 76% with a total editing efficiency of ≈20% in the two conserved sites. Collectively, this study demonstrates spatiotemporal liver genome editing as well as the feasibility of applying a nonviral CRISPR/Cas9 vector for HBV treatment, which may open up a new application for CRISPR therapeutics.

A Correlation Study of Liver Biopsy Appearances, Serum HBV DNA and HBsAg Quantification Levels of Nigerian Patients with Chronic Liver Disease

Background: Infection with the hepatitis B virus is highly endemic in much of sub-Saharan Africa where it remains an important cause of chronic ill-health. Quantitative assay of HBV DNA and HBsAg are emerging as useful tools in the management of chronic HBV infection. However, it is not yet clear how well each of these might accurately correlate with the extent of liver damage during the course of the disease. Nevertheless, liver biopsy, albeit an invasive test, affords a superior direct assessment of all stages of the disease. Objective: Thus, we set out to determine how well-correlated serum levels of HBV-DNA and/or HBsAg might be with each other and with the liver biopsy appearances. Methodology: This study was prospective and we enrolled, 83 new treatment-naïve patients, who met the study criteria. Each patient had liver biopsy assessments of the grade of necroinflammation (A) and stage of fibrosis (F), according to the METAVIR Scoring System. In addition, quantitative HBsAg serum levels by immunoassay and HBV DNA by real-time quantitative PCR protocols were made. The Spearman’s and Kruskal-Wallis statistical tests were employed to determine the correlations between these assays separately and with the respective histological grades and stages of liver biopsy. (p=0.05) Results: The greatest number of the patients were within the 21-40 year- age group. Using the Spearman rho’s statistical correlation test, we found a positive but weak correlation between the serum HBsAg and HBVDNA levels (p=0.075, r=0.198). The Kruskal-Wallis analysis revealed statistically significant correlation between HBV-DNA and the grade of necroinflammation but not with the stage of fibrosis. Conclusions: We found that the serum HBV DNA levels had the only statistically significant correlation with the liver histological disease. This relationship needs to be further examined in larger studies in the future.

Lower frequency of MDSCs was significantly related to functional cure in CHB patients treated with peginterferon.

Myeloid-derived suppressor cells (MDSCs) and CD4+ regulatory T cells (Tregs) expand during chronic hepatitis B virus (HBV) infection and inhibit antiviral immunity. However, the relationship between antiviral effect and the frequencies of those immune suppressive cells after pegylated interferon α-2a (PegIFNα-2a) therapy is not clearly understood. This study aimed to investigate the contribution of monocytic MDSCs (mMDSCs) and CD4+ Tregs to functional cure (HBsAg seroclearance) after PegIFNα-2a therapy and evaluate the effect of PegIFNα-2a therapy on these cells. Flow cytometry analysis was performed along with longitudinal immune monitoring of 97 hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) patients receiving PegIFNα-2a weekly for 48 weeks. The frequencies of mMDSCs and CD4+ Tregs increased in all HBV patients, and they were higher in the HBsAg persistence group than in the HBsAg seroclearance group. A significant decline in the frequency of mMDSCs was found in patients who realized functional cure after PegIFNα-2a treatment. In contrast, the frequency of CD4+ Tregs in both the HBsAg seroclearance and persistence groups significantly increased. Multivariate analyses indicated that the baseline serum HBsAg levels (p < .001) and mMDSCs frequency (p=.027) were independently associated with the HBsAg clearance, and the combined marker (HBsAg plus mMDSCs) displayed the highest specificity (93.1%) than any other markers in predicting HBsAg seroclearance. These results suggest that a poor response to PegIFNα-2a treatment in CHB patients may be related to the frequencies of immune suppressive cells, while the therapeutic targeting of these cells might be effective in boosting anti-HBV immunity.

Low anti-HBc levels are associated with lower risk of virological relapse after nucleos(t)ide analogue cessation in HBe antigen-negative patients.

Low anti-HBc serum levels at the time of therapy cessation were linked to a higher relapse risk in predominantly HBeAg-positive cohorts. We investigated the association of anti-HBc levels with relapse in HBeAg-negative patients. Serum levels of anti-HBc, HBsAg and HBcrAg were determined in 136 HBeAg-negative patients, participating in a vaccination trial (ABX-203, NCT02249988), before treatment cessation or vaccination. Importantly, vaccination showed no impact on relapse. The correlation between the biomarkers and their predictive value for relapse (HBV DNA >2000 IU/ml ± ALT >2xULN) was investigated. After therapy cessation 50% (N= 68) of patients relapsed. Median anti-HBc prior to treatment stop was significantly higher among relapsers compared to off-treatment responders (520 IU/ml vs. 330 IU/mL, p= .0098). The optimal anti-HBc cut-off to predict relapse was 325 IU/ml according to the Youden-Index. About 35% of patients with anti-HBc level < 325 IU/ml versus 60% of those with values ≥325 IU/mL relapsed (p= .0103; sensitivity 50%, specificity 75%). Combining the optimal cut-offs of HBsAg (>3008 IU/mL) or HBcrAg (≥1790 U/ml) with anti-HBc increased the proportion of patients with relapse to 80% (p< .0001) and 74% (p= .0006), respectively. In contrast to predominantly HBeAg-positive cohorts, in our cohort of HBeAg-negative patients lower anti-HBc levels are associated with a significantly lower relapse risk after nucleos(t)ide analogue cessation. The vast majority of included patients were either genotype B or C and the applicability to other genotypes has to be further evaluated. However, anti-HBc level as an indicator of the host response might be prospectively further explored for prediction models.

Randomised controlled trial: effect of metformin add-on therapy on functional cure in entecavir-treated patients with chronic hepatitis B

Introduction and ObjectivesHepatitis B surface antigen (HBsAg) clearance, indicating functional cure or resolved chronic hepatitis B (CHB), remains difficult to achieve via nucleos(t)ide analogue monotherapy. We investigated whether metformin add-on therapy could help achieve this goal in entecavir-treated patients with hepatitis B e antigen (HBeAg)-negative CHB. Patients and MethodsPatients with HBeAg-negative CHB who met eligibility criteria (entecavir treatment for > 12 months, HBsAg < 1000 IU/mL) were randomly assigned (1:1) to receive 24 weeks of either metformin (1000 mg, oral, once a day) or placebo (oral, once a day) add-on therapy. The group allocation was blinded for both patients and investigators. Efficacy and safety analyses were based on the intention-to-treat set. The primary outcome, serum HBsAg level (IU/mL) at weeks 24 and 36, was analysed using mixed models. ResultsSixty eligible patients were randomly assigned to the metformin (n = 29) and placebo (n = 31) groups. There was no substantial between-group difference in the HBsAg level at week 24 (adjusted mean difference 0.05, 95% confidence interval -0.04 to 0.13, p = 0.278) or week 36 (0.06, -0.03 to 0.15, p = 0.187), and no significant effect of group-by-time interaction on the HBsAg level throughout the trial (p = 0.814). The occurrence of total adverse events between the two groups was comparable (9 [31.0%] of 29 vs. 5 [16.1%] of 31, p = 0.227) and no patient experienced serious adverse events during the study. ConclusionAlthough it was safe, metformin add-on therapy did not accelerate HBsAg clearance in entecavir-treated patients with HBeAg-negative CHB.

Liver biopsy of chronic hepatitis B patients indicates HBV integration profile may complicate the endpoint and effect of entecavir treatment

AimsViral integration profiles attract increased interest in the study of HBV-related hepatocellular carcinoma (HCC), but their features in the early stage of infection and changes due to antiviral treatments remain largely unknown. MethodsLiver biopsies and paired blood samples were obtained from HBeAg-positive patients before and after 48 weeks of entecavir treatment, and a probe-based capture strategy was applied for analyzing the HBV integrations in these samples. Serum HBV markers, including viral DNA, pgRNA, and HBsAg, were longitudinally assessed. ResultsEntecavir treatment successfully reduced the levels of ALT, AST, and HBV serological markers (HBeAg, HBV pgRNA, and HBV DNA) in all patients (<40 years old). As expected, HBV integrations contributed to HBsAg production, with the total number of integrations positively correlated with serum HBsAg level (r = 0.47, P = 0.04). Along with repressed HBV replication, the number of viral integrations in liver biopsies decreased by about 1.94-fold after ETV treatment, with viral breakpoints significantly enriched within nt 1600–1900 of the HBV genome. No recurrent events were observed both at baseline and after treatment for the same individual, and only one same integration was found in two patients. Unlike in tumors, integrations in CHB biopsies seemed to have no chromosomal preference. Moreover, CHB integrations demonstrated lower enrichment scores for open active states than tumors, such as DNase, TssA, and ZNF/Rpts, and the scores reduced after ETV treatment. The antiviral therapy led to the disappearance of the enrichment tendency of integrations in both open chromatin and heterochromatin regions. ConclusionReduced HBV replications by the nucleoside analogue may lead to decreased viral integrations in the liver, and those contributing to the HBsAg production may consistently occur. The pattern of HBV integration after ETV treatment is more random and irregular, which may contribute to a reduced risk of liver cancer due to antiviral treatment.

Comparison of the Effect of Entecavir and Tenofovir on Serum HBsAg Levels in Chronic HBe-Negative Hepatitis B Patients

Background: The quantification of hepatitis B surface antigen (qHBsAg) was proposed as a helpful tool to monitor treatment efficacy with nucleos(t)ide analogs (NA) in HB e antigen-negative chronic HB. Objectives: The present study aimed to assess the effect of entecavir (ETV) and tenofovir dipivoxil (TDF) on qHBsAg kinetics and estimate the time necessary to achieve HBsAg clearance with each of these drugs. Methods: The study was conducted on 93 patients, 54 and 39 of whom were treated with ETV and TDF for a median time of 42 months, respectively. The qHBsAg was measured in 6-month intervals with the Elecsys HBsAg II Quantitative assay. The estimated time to undetectable HBsAg was calculated using the best-fitted curve analysis. Results: There was a significant decrease in qHBsAg titers in 79 (84.9%) patients with no difference between ETV and TDF groups (P = 0.754). The median quantitative HBsAg drop was 2003 IU/mL (interquartile range: 638.1 - 5010). The HBsAg levels decreased by 40.3 ± 25.9% on average. The expected time required for HBsAg clearance was comparable in both groups, equaling 104 and 114 months for TDF and ETV, respectively. Conclusions: The HBsAg clearance can be achieved in a substantial proportion of patients after additional 5 years of treatment with NA. The potency of TDF and ETV in qHBsAg reduction is similar.

Feasibility of immunotherapy in cancer patients with persistent or past hepatitis B or C virus infection.

Background and AimImmune checkpoint inhibitors (ICIs) can cause immune‐related adverse events in the liver. The risk of exacerbating liver injury is of concern in patients infected with hepatitis B virus (HBV) or hepatitis C virus (HCV), as immunotherapy can damage liver function because of the immune response against viral antigens. We assessed the feasibility of immunotherapy in HBV‐ or HCV‐infected patients.MethodsThis retrospective study included 266 patients with persistent or past HBV infection, 26 patients seropositive for anti‐HCV, and 820 patients with negative viral markers for HBV and HCV, who were treated with ICIs. ICI‐induced liver injury and changes in virological markers were analyzed.ResultsThe occurrence rates of ICI‐induced liver injury in the HBsAg‐positive, anti‐HBc‐positive/anti‐HBs‐positive, and anti‐HBc‐positive/anti‐HBs‐negative groups were 12.5, 21.6, and 19.1%, respectively, which were comparable with those of the negative for HBV‐ and HCV‐related markers group (20.9%). The frequency of any grade ICI‐induced liver injury was different among the HCV RNA‐positive (3/5; 60.0%), anti‐HCV‐positive/HCV RNA‐negative (2/21; 9.5%), and negative for HBV‐ and HCV‐related markers (171/820; 20.9%) groups (P = 0.045), with no significant difference in grade ≥2 ICI‐induced liver injury. In patients with persistent infection, neither serum HBV DNA, HBsAg, nor HCV RNA level changed significantly during ICI treatment. One of five treatment‐naïve HCV‐infected patients required interruption of ICI treatment due to virus‐related liver injury.ConclusionImmunotherapy is feasible for most cancer patients with chronic HBV or HCV infection; however, liver function and virological markers should be carefully monitored in treatment‐naïve patients, especially those with HCV infection, during ICI treatment.

Screening and Identification of HBV Epitopes Restricted by Multiple Prevalent HLA-A Allotypes.

Although host T cell immune responses to hepatitis B virus (HBV) have been demonstrated to have important influences on the outcome of HBV infection, the development of T cell epitope-based vaccine and T cell therapy and the clinical evaluation of specific T cell function are currently hampered markedly by the lack of validated HBV T cell epitopes covering broad patients. This study aimed to screen T cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and presenting by thirteen prevalent human leukocyte antigen (HLA)-A allotypes which gather a total gene frequency of around 95% in China and Northeast Asia populations. 187 epitopes were in silico predicted. Of which, 62 epitopes were then functionally validated as real-world HBV T cell epitopes by ex vivo IFN-γ ELISPOT assay and in vitro co-cultures using peripheral blood mononuclear cells (PBMCs) from HBV infected patients. Furthermore, the HLA-A cross-restrictions of each epitope were identified by peptide competitive binding assay using transfected HMy2.CIR cell lines, and by HLA-A/peptide docking as well as molecular dynamic simulation. Finally, a peptide library containing 105 validated epitopes which cross-binding by 13 prevalent HLA-A allotypes were used in ELISPOT assay to enumerate HBV-specific T cells for 116 patients with HBV infection. The spot forming units (SFUs) was significantly correlated with serum HBsAg level as confirmed by multivariate linear regression analysis. This study functionally validated 62 T cell epitopes from HBV main proteins and elucidated their HLA-A restrictions and provided an alternative ELISPOT assay using validated epitope peptides rather than conventional overlapping peptides for the clinical evaluation of HBV-specific T cell responses.

Observation of PD-1+CXCR5+CD4+T lymphocyte and sPD-1 levels in HBeAg positive chronic hepatitis B virus carriers treated with entecavir

Objective: To dynamically observe the clinical efficacy of entecavir and the changes of PD-1+CXCR5+CD4+T lymphocytes and sPD-1 levels in peripheral blood of HBeAg-positive chronic hepatitis B virus carriers treated with entecavir, and further explore its clinical significance. Methods: There were 31 cases of chronic hepatitis B virus carriers in the treatment group (A), 32 cases of chronic hepatitis B virus carriers in the treatment group (B), and 15 cases of chronic hepatitis B virus carriers in the non-treatment group (C).Three groups peripheral blood samples and clinical data at 0, 24 and 48 weeks were collected and compared. PD-1+CXCR5+CD4+T lymphocytes were detected by flow cytometry, and the level of sPD-1 was detected by enzyme-linked immunosorbent assay. ANOVA and Spearman correlation analysis were performed on the measurement data among the three groups. Results: At week 0, the serum levels of HBsAg, HBeAg and HBV DNA were significantly higher in groups A and C than group B. PD-1+CXCR5+CD4+T lymphocytes in peripheral blood were significantly higher in group B (4.70%±1.58%) than group A (3.25%±1.01%) and group C (2.77%±0.67%) (F=16.65, P<0.05). There was no significant difference between group A and group C (P>0.05). Peripheral blood sPD-1 in group B [(1 866.62±1 472.70) pg/ml] was significantly higher than group A [(824.86±538.66) pg/ml] and group C [(618.19±602.62) pg/ml] (F=10.95, P<0.05). There was no significant difference between group A and group C (P>0.05). At 48 weeks, the serum HBsAg did not decrease significantly in groups A and C than baseline (P>0.05), but were significantly higher than group B (P<0.05). Serum HBeAg levels were decreased significantly in groups A and B than baseline (P<0.05). <0.05), but group A was significantly higher than group B (P<0.05), and there was no significant difference between group A and group C (P>0.05). Serum HBV DNA level was significantly lower in groups A and B than group C (P<0.05), and there was no significant difference between group A and group B (P>0.05). Peripheral blood PD-1+CXCR5+CD4+T lymphocytes were significantly lower in Group A (1.56%±0.73%) and group B (1.32%±0.43%) than group C (2.64%±0.85%) (P<0.05). Peripheral blood sPD-1 were significantly lower in group A [(289.05±215.86) pg/ml] and group B [(236.01±173.92) pg/ml] than group C [(650.34±598.46) pg/ml] (P<0.05). There was no significant difference between group A and group B. Correlation analysis results: In group A at 48 weeks, the decreased level of PD-1+CXCR5+CD4+T lymphocyte ratio had no correlation with the decreased level of HBsAg and HBV DNA, but was positively correlated with the decreased level of HBeAg (r=0.376, P<0.05). The decreased level of sPD-1 had no correlation with the changes of HBsAg, but was positively correlated with the decreased levels of HBeAg and HBV DNA (r=0.598 and 0.384, P<0.05). In group B at 48 weeks, the decreased levels of PD-1+CXCR5+CD4+T lymphocytes and sPD-1 were positively correlated with the decreased levels of HBsAg, HBeAg, and HBV DNA (P<0.05). Conclusion: Hepatitis B virus replication and expressions in HBeAg-positive chronic hepatitis B virus carriers were significantly inhibited after 48 weeks of antiviral treatment, which is related not only to entecavir treatment, but also to the immunological mechanism involved in sPD-1. Moreover, the inhibition of HBeAg expression is associated with a decrease in the number and/or activity of PD-1+CXCR5+CD4+T lymphocytes.

Serum HBV RNA predicts HBeAg clearance and seroconversion in patients with chronic hepatitis B treated with nucleos(t)ide analogues.

This study evaluated the predictive value of serum HBV DNA, HBV RNA, HBcrAg, HBsAg, intrahepatic HBV DNA and cccDNA for HBeAg clearance and seroconversion during long‐term treatment with nucleos(t)ide analogues (NAs) in patients with chronic hepatitis B (CHB). A single centre, prospective cohort of CHB patients was used for this study. Serum HBV RNA levels were retrospectively measured at baseline, 6, 12, 24, 36, 48, 60, 72 and 84 months post‐NAs treatment. Serum HBsAg and HBcrAg levels were quantified at baseline, month 6, 60 and 72. Histological samples from liver biopsy at baseline and month 60 were analysed for intrahepatic HBV DNA and cccDNA. Eighty‐three HBeAg‐positive patients were enrolled with a median follow‐up time of 108 months (range 18–138 months). Of them, 53 (63.86%) patients achieved HBeAg clearance, and 37 (44.58%) achieved HBeAg seroconversion. Cox multivariate analysis showed that only baseline HBV RNA was independently associated with HBeAg clearance and seroconversion (<5.45 log10copies/mL, HR = 5.06, 95% CI: 1.87–13.71, p = .001; HR = 3.38, 95% CI: 1.28–8.91, p = .01). The independent association with HBeAg clearance and seroconversion remained for HBV RNA levels at month 6 (<4.72 log10copies/mL, HR = 4.16, 95% CI: 1.61–10.72, p = .003; HR = 6.52, 95% CI: 1.85–22.94, p = .003) and month 12 (<4.08 log10 copies/mL, HR = 3.68, 95% CI: 1.96–6.90, p < .001; HR = 2.79, 95% CI: 1.31–5.94, p = .008). The AUCs of baseline HBV RNA for predicting the HBeAg clearance (0.83, 95% CI: 0.70–0.96, 0.83, 95% CI: 0.70–0.96 and 0.82, 95% CI: 0.69–0.95 respectively) and seroconversion (0.89, 95% CI: 0.77–1.00; 0.81, 95% CI: 0.66–0.95 and 0.84, 95% CI: 0.71–0.98 respectively) at month 36, 60 and 84 were higher than those of HBV DNA, HBsAg and HBcrAg. In conclusion, lower serum HBV RNA at baseline, month 6 and 12 post‐NAs treatment could predict HBeAg clearance and seroconversion during long‐term NAs treatment.

Dynamics of Serum Pregenome RNA in Chronic Hepatitis B Patients Receiving 96-Month Nucleos(t)ide Analog Therapy

BackgroundTissue covalently closed circular DNA (cccDNA) can reflect the activity of HBV replication. However, it is impractical to assess intrahepatic cccDNA in every outpatient. Serum pregenome RNA (pgRNA) is transcribed from intrahepatic cccDNA and may reflect the activity of intrahepatic cccDNA. We explored the dynamics and the potential role of serum pgRNA in patients receiving long-term NAs treatment.MethodsSerum pgRNA, HBV DNA, HBsAg, HBeAg, and ALT levels were quantified, and the relationships between serum pgRNA and these common clinical indicators before and after the treatment were investigated.ResultsSerum pgRNA showed dynamic change during the 96-month NAs therapy, and serum pgRNA levels were positive and detectable in 19 patients with undetectable serum HBV DNA. Serum pgRNA showed strong and positive correlation with serum HBV DNA (r = 0.693, p < 0.001) and serum HBsAg levels (r = 0.621, p < 0.001) at baseline. Patients with HBeAg seroconversion had lower baseline serum pgRNA levels (p = 0.002). The area under the curve (AUC) of baseline serum pgRNA for predicting HBeAg seroconversion was 0.742 (95% CI: 0.606–0.850) with 63.16% sensitivity and 80.56% specificity. The cumulative HBeAg seroconversion rate was higher in patients with low serum pgRNA (p = 0.001).ConclusionSerum pgRNA of low level at baseline or great decline at month 6 may independently predict the high incidence of undetectable serum pgRNA at year 4 following NAs therapy, and the baseline serum pgRNA may serve as a novel predictor for HBeAg seroconversion during NAs therapy.

Human hepatocyte-enriched miRNA-192-3p promotes HBV replication through inhibiting Akt/mTOR signalling by targeting ZNF143 in hepatic cell lines

ABSTRACT Previous studies have revealed multiple tissue- or cell-specific or enriched miRNA profiles. However, miRNA profiles enriched in hepatic cell types and their effect on HBV replication have not been well elucidated. In this study, primary human hepatocytes (PHHs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) were prepared from liver specimens of non-HBV-infected patients. Four hepatic cell type-enriched miRNA profiles were identified from purified liver cells miRNA microarray assay. The results revealed that 12 miRNAs, including miR-122-5p and miR-192-3p were PHH-enriched; 9 miRNAs, including miR-142-5p and miR-155-5p were KC-enriched; 6 miRNAs, including miR-126-3p and miR-222-3p were LSEC-enriched; and 14 miRNAs, including miR-214-3p and miR-199a-3p were HSC-enriched. By testing the effect of 11 PHH-enriched miRNAs on HBV production, we observed that miR-192-3p had the greatest pro-virus effect in hepatic cell lines. Moreover, we further found that miR-192-3p promoted HBV replication and gene expression through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in HepG2.2.15 cells. Additionally, the serum and hepatic miR-192-3p expression levels were significantly higher in chronic hepatitis B patients than in healthy controls and serum miR-192-3p positively correlated with the serum levels of HBV DNA and HBsAg. Collectively, we identified miRNA profiles enriched in four hepatic cell types and revealed that PHH-enriched miR-192-3p promoted HBV replication through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in hepatic cell lines. Our study provides a specific perspective for the role of hepatic cell type-enriched miRNA in interaction with viral replication and various liver pathogenesis.

Quantitative HBsAg an unreliable marker for diagnosis and disease progression in genotype F chronic HBeAg-negative infections.

Quantitative hepatitis B surface antigen (qHBsAg) has been proposed as a biomarker to distinguish HBeAg-negative chronic infections (ENI) from HBeAg-negative chronic hepatitis (ENH), identify patients prone to achieving sustained HBsAg loss, and predict the risk of liver disease progression. There is evidence that qHBsAg varies among genotypes, however there is a paucity of data on genotype F. The aim of this study was to investigate the performance of qHBsAg in the diagnosis and evolution of genotype F chronic HBeAg-negative infections. HBV-DNA and HBsAg levels from 153 patients with ENI were correlated with the genotype. Liver disease progression was assessed by abdominal ultrasound and a transient elastography. The qHBsAg levels were significantly different among genotypes (p <0.001), being GTF: 4.0±1.1, GTA: 3.9±0.6 and GTD: 2.4±0.9 Log10 IU/ml. Only 10.7 and 11.5% of GTA and GTF showed qHBsAg <3.0 Log10 IU/ml. Regardless of HBV genotype, HBsAg clearance was observed in 17 cases, of which 12 showed qHBsAg <100 IU/ml before clearance. Despite of a large number of patients having qHBsAg >3.0 log10 IU/ml, no cases of advanced liver disease were observed at the end of follow-up. This study provides new insights into the impact of HBV genotypes, in particular GTF, on serum HBsAg levels, emphasizing the need to implement a genotype-specific cut-off to achieve diagnostic certainty in the identification of ENI and the risk of liver disease progression. Regardless of HBV genotype, qHBsAg has been shown to be a powerful and reliable biomarker for predicting HBsAg loss.

Superiority of tenofovir alafenamide fumarate over entecavir for serum HBsAg level reduction in patients with chronic HBV infection: A 144-week outcome study after switching of the nucleos(t)ide analog.

BackgroundTo evaluate the long-term efficacy of switching of the nucleos(t)ide analog used for treatment from entecavir (ETV) to tenofovir alafenamide fumarate (TAF) in patients with chronic HBV infection.MethodsA total of 103 patients with serum HBsAg levels of ≥100 IU/mL who had received ETV were enrolled. The nucleos(t)ide analog used for the treatment was switched from ETV to TAF, and the changes in serum HBsAg levels during the 144-week period before and after the drug switching were compared in 74 patients who had received ETV at least for 192 weeks.ResultsSignificant decreases of serum HBsAg levels were observed during both the ETV and the TAF administration period, although the degree of reduction was greater during the latter period than during the former period (P<0.001). Significant decreases of serum HBsAg levels were seen in both patients with genotype B HBV infection and genotype C HBV infection, irrespective of the serum HBsAg and HBcrAg levels at the time of the drug switching.ConclusionSwitching of the nucleos(t)ide analog used for treatment from ETV to TAF merits consideration in patients with chronic HBV infection, since the extent of reduction of the serum HBsAg level was greater during the TAF treatment period than during the ETV treatment period.

Use of a Deep Learning Approach for the Sensitive Prediction of Hepatitis B Surface Antigen Levels in Inactive Carrier Patients.

Deep learning is a subset of machine learning that can be employed to accurately predict biological transitions. Eliminating hepatitis B surface antigens (HBsAgs) is the final therapeutic endpoint for chronic hepatitis B. Reliable predictors of the disappearance or reduction in HBsAg levels have not been established. Accurate predictions are vital to successful treatment, and corresponding efforts are ongoing worldwide. Therefore, this study aimed to identify an optimal deep learning model to predict the changes in HBsAg levels in daily clinical practice for inactive carrier patients. We identified patients whose HBsAg levels were evaluated over 10 years. The results of routine liver biochemical function tests, including serum HBsAg levels for 1, 2, 5, and 10 years, and biometric information were obtained. Data of 90 patients were included for adaptive training. The predictive models were built based on algorithms set up by SONY Neural Network Console, and their accuracy was compared using statistical analysis. Multiple regression analysis revealed a mean absolute percentage error of 58%, and deep learning revealed a mean absolute percentage error of 15%; thus, deep learning is an accurate predictive discriminant tool. This study demonstrated the potential of deep learning algorithms to predict clinical outcomes.

The Relationship Between Collagen Proportionate Area and Hepatitis B Surface Antigen Levels in E Antigen Positive Hepatitis B Cirrhosis.

Quantitative serum hepatitis B surface antigen (HbsAg) has been widely used as a biomarker for treatment response and prognosis in chronic hepatitis B infection, and has recently been found associated with liver histology in e-antigen positive patients. A histological measurement as a continuous variable-collagen proportionate area (CPA)-is appropriate to assess liver fibrosis degree and substages cirrhosis. We, therefore, aimed to explore the association between serum quantitative HBsAg and CPA in e antigenpositive hepatitis B cirrhosis. Liver fibrosis staging was evaluated by METAVIR semiquantitative scoring system, only patients with METAVIR fibrosis stage 4 were included. All liver sections were stained with picroSirius red for determination of collagen quantification by digital image analysis. Mean CPA value was 23.46%. The percentage of patients with different classification of CPA (30%) were 25.8%, 57.8%, and 16.4%, respectively. A modest correlation was found between CPA and serum HBsAg level (r = -0.306, P =.001). Hepatitis B surface antigen level is independently associated with CPA in multivariable linear regression analyses. Serum HBsAg levels can predict liver fibrosis determined by CPA in HBeAg-positive hepatitis B cirrhosis.