Sera of Individuals Chronically Infected with Hepatitis B Virus (HBV) Contain Diverse RNA Types Produced by HBV Replication or Derived from Integrated HBV DNA.

This study aimed to better characterize the repertoire of serum hepatitis B virus (HBV) RNAs during chronic HBV infection in humans, which remains understudied. Using reverse transcription-PCR (RT-PCR), real-time quantitative PCR (RT-qPCR), RNA-sequencing, and immunoprecipitation, we found that (i) >50% of serum samples bore different amounts of HBV replication-derived RNAs (rd-RNAs); (ii) a few samples contained RNAs transcribed from integrated HBV DNA, including 5'-HBV-human-3' RNAs (integrant-derived RNAs [id-RNAs]) and 5'-human-HBV-3' transcripts, as a minority of serum HBV RNAs; (iii) spliced HBV RNAs were abundant in <50% of analyzed samples; (iv) most serum rd-RNAs were polyadenylated via conventional HBV polyadenylation signal; (v) pregenomic RNA (pgRNA) was the major component of the pool of serum RNAs; (vi) the area of HBV positions 1531 to 1739 had very high RNA read coverage and thus should be used as a target for detecting serum HBV RNAs; (vii) the vast majority of rd-RNAs and pgRNA were associated with HBV virions but not with unenveloped capsids, exosomes, classic microvesicles, or apoptotic vesicles and bodies; (viii) considerable rd-RNAs presence in the circulating immune complexes was found in a few samples; and (ix) serum relaxed circular DNA (rcDNA) and rd-RNAs should be quantified simultaneously to evaluate HBV replication status and efficacy of anti-HBV therapy with nucleos(t)ide analogs. In summary, sera contain various HBV RNA types of different origin, which are likely secreted via different mechanisms. In addition, since we previously showed that id-RNAs were abundant or predominant HBV RNAs in many of liver and hepatocellular carcinoma tissues as compared to rd-RNAs, there is likely a mechanism favoring the egress of replication-derived RNAs. IMPORTANCE The presence of integrant-derived RNAs (id-RNAs) and 5'-human-HBV-3' transcripts derived from integrated hepatitis B virus (HBV) DNA in sera was demonstrated for the first time. Thus, sera of individuals chronically infected with HBV contained both replication-derived and integrant-transcribed HBV RNAs. The majority of serum HBV RNAs were the transcripts produced by HBV genome replication, which were associated with HBV virions and not with other types of extracellular vesicles. These and other above-mentioned findings advanced our understanding of the HBV life cycle. In addition, the study suggested a promising target area on the HBV genome to increase sensitivity of the detection of serum HBV RNAs and supported the idea that simultaneous detection of replication-derived RNAs (rd-RNAs) and relaxed circular DNA (rcDNA) in serum provides more adequate evaluation of (i) the HBV genome replication status and (ii) the durability and efficiency of the therapy with anti-HBV nucleos(t)ide analogs, which could be useful for improvement of the diagnostics and treatment of HBV-infected individuals.

[A real-world study on the features of postpartum hepatitis flares in pregnant women with chronic HBV infection].

Objective: To analyze the clinical features of postpartum hepatitis flares in pregnant women with hepatitis B virus (HBV) infection. Methods: A retrospective study was conducted. Patients who met the enrollment criteria were included. Liver function and HBV virology tests were collected from pregnant women with chronic HBV infection at delivery, 6, 24, 36, and 48 weeks after delivery through the hospital information and test system. Additionally, antiviral therapy types and drug withdrawal times were collected. Statistical analysis was performed on all the resulting data. Results: A total of 533 pregnant women who met the inclusion criteria were included, with all patients aged (29.5±3.7) years old. A total of 408 cases received antiviral drugs during pregnancy to interrupt mother-to-child transmission. There was no significant difference in the levels of alanine aminotransferase (ALT, z = -1.981, P = 0.048), aspartate aminotransferase (AST, z = -3.956, P < 0.001), HBV load (z = -15.292, P < 0.001), and HBeAg (z = -4.77, P < 0.001) at delivery in patients who received medication and those who did not. All patients ALT, AST, total bilirubin, direct bilirubin, and albumin showed an upward trend within six weeks after delivery. A total of 231 cases developed hepatitis within 48 weeks after delivery. Among them, 173 cases first showed ALT abnormalities within six weeks postpartum. Conclusion: Hepatitis flare incidence peaked six weeks after delivery or six weeks after drug withdrawal in pregnant women with chronic HBV infection.

The Role of ALPK1 in Inhibiting Hepatitis B Virus Replication Facilitates the Identification of ALPK1 P660L Variant for Predicting Response to Pegylated Interferon α Therapy.

BACKGROUND: Alpha kinase 1 (ALPK1) agonist has recently been reported to demonstrate anti-hepatitis B virus (HBV) efficacy via activating NF-κB signaling, which is crucial for maximizing interferon (IFN) responses. Here, we investigated the impact of ALPK1 on HBV replication and explored ALPK1 variants for predicting the response to pegylated IFN-α (PegIFN-α) treatment. METHODS: The potential anti-HBV effect of ALPK1 was evaluated in HBV-integrated and HBV-infected hepatoma cells. The potentially functional genetic variants of ALPK1 were screened out, and their correlations with PegIFN-α treatment response were assessed in 945 hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B (CHB). RESULTS: We revealed that ALPK1 inhibited HBV replication in hepatocytes via activating the JAK-STAT pathway. ALPK1 overexpression improved the anti-HBV effect of IFN-α in cell models. A missense variant, rs35389530 (P660L), of ALPK1 was strongly associated with combined response (CR; namely, HBeAg seroconversion and HBV DNA level <3.3log10 IU/mL) to PegIFN-α treatment in patients with CHB (P = 2.12 × 10-6). Moreover, a polygenic score integrating ALPK1_rs35389530 and 2 additional genetic variants was further significantly associated with CR (Ptrend = 9.28 × 10-7), hepatitis B surface antigen (HBsAg) level (Ptrend = .0002), and HBsAg loss (Ptrend = .025). CONCLUSIONS: The anti-HBV effects of ALPK1 through activating JAK-STAT pathway provides a new perspective for CHB therapy. ALPK1_rs35389530 and polygenic score are potential biomarkers to predict PegIFN-α treatment response and may be used for optimizing CHB treatment.

Characteristics of Older Patients With Immunotolerant Chronic Hepatitis B Virus Infection.

BACKGROUND & AIMS: Most patients in the immunotolerant (IT) phase of chronic hepatitis B (CHB) transition to the immune active (IA-hepatitis B surface antigen [HBeAg]+) phase by early adulthood. We examined characteristics of adults in the IT vs IA-HBeAg+ phase and rate of transition from IT to other phases of CHB, with a focus on those ≥40 years. METHODS: Demographic, clinical, and virologic characteristics of participants in the Hepatitis B Research Network adult cohort study with IT CHB (alanine aminotransferase ≤1.5 × upper limit of normal, hepatitis B virus DNA >107 IU/mL) were compared by age category, and to those with IA-HBeAg+ CHB in cross-sectional analysis. This study received institutional review board approval at all participating centers. RESULTS: Of 107 adult IT participants, 52 (48%) were <30, 33 (31%) were 30 to 39, and 22 (21%) were ≥40 years old (maximum, 71 years). Among IT groups, the proportion born in Asia and duration of CHB were greater in older IT groups, but virologic and liver disease characteristics were similar. Compared with IA-HBeAg+ participants (n = 192), IT participants were younger, fewer were men, more were Asian, and platelets, qHBsAg, and qHBeAg levels were higher. Similar differences were observed when comparisons were made with the ≥40 years IT group. Among IT participants, 60 (56%) transitioned during 206 person-years of follow-up. The phase transition rate per 100 person-years was highest in the <30 years group (33.0 [95% confidence interval [CI], 23.4-46.7]) vs the 30 to 39 years group (24.8 [95% CI, 15.6-39.4]) and ≥40 group (27.4 [95% CI, 14.8-50.9]), but 95% CIs overlapped. CONCLUSIONS: In a large North American population, over 50% of adults in the IT phase of CHB were ≥30 years and 20% were ≥40 years old, but older IT patients had similar characteristics and rates of transition as younger IT patients.

Incidence and factors influencing sleep disorders in patients with chronic hepatitis B infection: A case-control study.

Chronic hepatitis B (HBV) infection is a disease that imposes a considerable financial burden on patients and can lead to sleep disorders (SDs), resulting in a serious deterioration to patient quality of life. This study aimed to determine the prevalence of SDs in patients with HBV and the correlated sociodemographic and clinical characteristics. A total of 747 patients with chronic HBV infection were recruited. All patients completed the Pittsburgh Sleep Quality Index (PSQI), Patient Health Questionnaire-9 (PHQ-9), Generalized Anxiety Disorder-7 scale, Social Support Rating Scale, Short Form 36 Health Survey (SF-36), and Visual Analogue Scale (VAS). The total PSQI score of patients with each type of chronic HBV infection was significantly higher compared to healthy Chinese adults (p < .05). The incidence of SDs in HBV carriers and patients with mild HBV, moderate HBV, severe HBV, liver failure, compensated cirrhosis, and decompensated liver cirrhosis was 25%, 26%, 32%, 47%, 56%, 31%, and 49%, respectively. The incidence of SDs in all patients with chronic HBV infection was 30%. Binary logistic regression analysis revealed that the course of disease, aspartate aminotransferase levels, PHQ-9 scores, and VAS scores were independent risk factors for SDs, while the total SF-36 score was a protective factor for SDs (all p < .05). In conclusion, the prevalence of SDs was significantly higher in patients with chronic hepatitis B compared to healthy subjects. The independent risk factors for SDs included disease duration, aspartate aminotransferase levels, depression, and fatigue. Clinicians should pay more attention to SDs in patients with chronic HBV infection.

Development a novel nomogram model for predicting significant hepatic histological changes in chronic hepatitis B virus carriers.

A proportion of chronic hepatitis B virus (HBV) carriers with normal alanine transaminase (ALT) present with significant liver histological changes (SLHC). To construct a noninvasive nomogram model to identify SLHC in chronic HBV carriers with different upper limits of normal (ULNs) for ALT. The training cohort consisted of 732 chronic HBV carriers who were stratified into four sets according to different ULNs for ALT: chronic HBV carriers I, II, III, and IV. The external validation cohort comprised 277 chronic HBV carriers. Logistic regression and least absolute shrinkage and selection operator analyses were applied to develop a nomogram model to predict SLHC. A nomogram model-HBGP (based on hepatitis B surface antigen, gamma-glutamyl transpeptidase, and platelet count) demonstrated good performance in diagnosing SLHC with area under the curve (AUCs) of 0.866 (95% confidence interval [CI]: 0.839-0.892) and 0.885 (95% CI: 0.845-0.925) in the training and validation cohorts, respectively. Furthermore, HBGP displayed high diagnostic values for SLHC with AUCs of 0.866 (95% CI: 0.839-0.892), 0.868 (95% CI: 0.838-0.898), 0.865 (95% CI: 0.828-0.901), and 0.853 (95% CI: 0.798-0.908) in chronic HBV carriers I, II, III, and IV, respectively. Additionally, HBGP showed greater ability in predicting SLHC compared with the existing predictors. HBGP has shown high predictive performance for SLHC, and thus may lead to an informed decision on the initiation of antiviral treatment.

Fatty liver is associated with significant liver inflammation and increases the burden of advanced fibrosis in chronic HBV infection.

BACKGROUND: Concurrent non-alcoholic fatty liver disease (NAFLD) is common in patients with chronic HBV infection. But the impact of fatty liver on the histologic progression of HBV infection remains controversial. METHODS: Consecutive HBV-infected patients who underwent liver biopsy between 2016 and 2021 were included. Alcohol consumption and other types of viral hepatitis were excluded. All biopsies were scored for grading and staging by Scheuer's score, and the steatosis was scored as an estimate of the percentage of liver parenchyma replaced by fat. Logistic regression analyses were applied to assess the associated factors for significant liver inflammation (G ≥ 2), significant fibrosis (S ≥ 2) and advanced fibrosis (S ≥ 3). RESULTS: Among the 871 HBV-infected patients, hepatic steatosis was prevalent in 255 patients (29.28%). Significant liver inflammation was present in 461 patients (52.93%). Significant fibrosis was observed in 527 patients (60.51%), while advanced liver fibrosis was observed in 171 patients (19.63%). Patients with concomitant NAFLD were more likely to have significant liver inflammation and advanced fibrosis. Fatty liver was an independent risk factor for significant liver inflammation (OR: 2.117, 95% CI: 1.500-2.988), but it could not predict the development of fibrosis. Especially, in HBV-infected patients with persistent normal ALT (immune tolerant and inactive carrier phase), the presence of significant liver inflammation was higher in NAFLD than those without NAFLD. The prevalence of advanced liver fibrosis was higher in NAFLD than non-NAFLD only in the immune tolerant phase, while NAFLD did not increase fibrosis burden in other stages of HBV infection. We developed a predictive model for significant liver inflammation with the area under receiver operating characteristic curve (AUROC) of 0.825, and a model for significant fibrosis with the AUROC of 0.760. CONCLUSIONS: NAFLD is independently associated with significant liver inflammation, and increases the burden of advanced liver fibrosis in HBV-infected patients. The influence of NAFLD on the degree of liver inflammation and fibrosis is different in distinct clinical phases of chronic HBV infection.

Unusual serological profile in hepatitis B virus (HBV) infection associated with a probable clinical case of acute exacerbation of pre-existing chronic HBV infection.

BACKGROUND: Acute or chronic HBV infection in an individual can be laboratory characterized according to the serological profile of the viral markers in the bloodstream, and the dynamics monitoring of these markers is necessary to assess the disorder course and the infection outcome. However, under certain circumstances unusual or atypical serological profiles may be observed in both acute and chronic HBV infection. They are considered as such because they do not properly characterize the form or infection clinical phase or because they seem inconsistent, considering the viral markers dynamics in both clinical contexts. This manuscript comprises the analysis of an unusual serological profile in HBV infection. METHODS AND RESULTS: This clinical-laboratory study, had as reference a patient who presented clinical profile suggestive of acute HBV infection after recent exposure, whose laboratory data were initially compatible with this clinical presentation. However, the serological profile analysis and its monitoring demonstrated unusual pattern of viral markers expression, which has been observed in several clinical contexts, and is often associated a number of agent- or host-related factors. CONCLUSION: The serological profile analyzed here, associated with the biochemical markers serum levels found, is indicative of active chronic infection, consequence of viral reactivation. This finding suggests that in the event of unusual serological profiles in HBV infection, if the influence of agent- or host-related factors is not properly considered and neither the viral markers dynamics properly analyzed, there may be mistake in the infection clinical diagnosis, especially when the patient's clinical and epidemiological history is unknown.

Droplet digital PCR assay as an innovative and promising highly sensitive assay to unveil residual and cryptic HBV replication in peripheral compartment.

Droplet digital PCR is an innovative and promising approach for highly sensitive quantification of nucleic acids that is being increasingly used in the field of clinical virology, including the setting of hepatitis B virus (HBV). Here, we comprehensively report a robust and reproducible ddPCR assay for the highly sensitive quantification of serum HBV-DNA. The assay showed a limit of detection of 4 copies/ml (<1IU/ml) by Probit analysis, showed a good linearity (R2 = 0.94) and a high intra- and inter-run reproducibility with differences between the values obtained in the same run or in two independent runs never exceeding 0.14logcopies/mL and 0.21logcopies/mL, respectively. By analysing serum samples from chronically HBV infected patients (mostly under antiviral treatment), ddPCR successfully quantified serum HBV-DNA in 89.8% of patients with detectable serum HBV-DNA < 20 IU/mL [equivalent to <112copies/ml] by classical Real-Time PCR assay, with a median (IQR) of 8(5-14)IU/mL [45(28-78)copies/ml], and in 66.7% of patients with undetectable serum HBV-DNA, with a median (IQR) of 5(4-9)IU/mL [28(20-50)copies/ml]. Similarly, by analysing serum samples from patients with a serological profile compatible with occult HBV infection (anti-HBc+/HBsAg-), ddPCR successfully quantified serum HBV-DNA in 40% of patients with a median (IQR) value of 1(1-2)IU/mL [5(5-11)copies/ml], in line with the extremely limited viral replication typically observed in occult HBV infection. Overall, the availability of assays for the highly sensitive quantification of serum HBV-DNA can provide an added value in optimizing the diagnosis of occult hepatitis B infection, improving the therapeutic management of chronically HBV infected patients, also in the light of innovative drugs (upcoming in clinical practise) aimed at achieving HBV functional cure.

Differential gene expression, irrespective of circulating Hepatitis B Surface Antigen levels, between Inactive Carrier and Nucleos(t)ide Analogue-Treated Hepatitis B Virus patients.

Long-term viremia control in chronic HBV patients occurs either spontaneously in inactive carrier (IC) patients or therapy-induced by nucleos(t)ide analogues (NUC). To better understand the characteristics of viremia control, we evaluated gene expression in purified leukocyte subsets from IC versus NUC-treated patients, and evaluated the putative modulatory effects of hepatitis B surface antigen (HBsAg). We observed that gene expression in NUC-treated patients differed markedly from IC patients, especially in dendritic cells, monocytes, and CD8+ T cells, while serum HBsAg levels had little effect. Nevertheless, based on our findings it cannot be excluded that HBsAg may act locally in the infected liver or preferentially affects HBV-specific cells.

Hepatitis B core-related antigen serum levels are associated with significant liver fibrosis in treatment-naive chronic HBV infection patients.

Several hepatitis B virus (HBV) serum markers have been identified as risk factors for liver fibrosis in patients with chronic HBV infection, and several noninvasive fibrosis tests based on serum indexes are now used to identify the severity of liver fibrosis. We aimed to identify the relationship between hepatitis B core-related antigen (HBcrAg) serum levels and liver fibrosis in treatment-naive chronic HBV infection patients. A total of 246 treatment-naive chronic HBV infection patients were enrolled in this study. All of the patients underwent liver biopsies at baseline. Using the METAVIR fibrosis stages, there were 15, 140, 50, 26 and 15 patients in the F0, F1, F2, F3 and F4 stages (METAVIR scoring system), respectively. The biochemical, serological and virological parameters were measured using standard laboratory procedures. The HBcrAg serum levels of the patients were examined via ELISA. HBcrAg serum levels of F2, F3 and F4 stage patients were significantly higher than those of nonsignificant liver fibrosis patients (METAVIR F0-F1), but there were no significant differences among F2, F3 and F4 stage patients. Serum HBcrAg (OR, 2.18; 95% confidence interval [CI], 1.51-3.16), albumin (ALB) (OR, 0.60; 95% CI, 0.41-0.87), prothrombin activity (PTA) (OR, 0.58; 95% CI, 0.40-0.83) and platelet (PLT) counts (OR, 0.38; 95% CI, 0.25-0.57) were associated with significant liver fibrosis (METAVIR F2-F4). The serum HBcrAg value enabled the correct identification of patients with significant fibrosis, with an area under the receiver operating characteristic curve of 0.81 (95% CI, 0.75-0.88). The APRI, FIB-4 index and ALBI score can identify significant liver fibrosis with an area under the ROC curve of 0.74 (95% CI, 0.66-0.81), 0.73 (95% CI, 0.65-0.80) and 0.63 (95% CI, 0.55-0.72), respectively. Compared with these three indexes, the accuracy rate of diagnosis of significant fibrosis based on HBcrAg was higher than that of the FIB-4 index (p = 0.0479) and ALBI score (p = 0.0030). HBcrAg, ALB, PTA serum levels and PLT counts were associated with significant liver fibrosis in treatment-naive chronic HBV infection patients. HBcrAg serum levels enabled the correct identification of patients with significant fibrosis (METAVIR F2-F4), and HBcrAg was more effective than the FIB-4 index and ALBI score.

Signal transducer and activator of transcription 3 cooperates with androgen receptor/cell cycle-related kinase signalling pathway in the progression of hepatitis B virus infection and gender differences.

The study aimed to investigate the role of androgen receptor (AR)/cell cycle-related kinase (CCRK) signalling pathway in chronic hepatitis B virus (HBV) infection and gender differences, and the contribution of AR regulatory factor signal transducer and activator of transcription 3 (STAT3) in it. AR, CCRK, and phosphorylated STAT3 expressions in liver tissues of chronic HBV-infected patients and non-HBV controls were determined by western blot and compared between genders. The relationships of expression levels with serum HBV DNA levels, liver inflammation activity, and fibrosis score were analysed in chronic HBV-infected patients. The relationships between expression levels of three proteins were also analysed. HBV-infected patients had significantly higher expression levels of AR, CCRK, and p-STAT3Tyr705 compared with controls (p < .01). The expression levels of AR, CCRK, and p-STAT3Tyr705 in chronic HBV-infected patients with severe inflammation were significantly higher than those with mild inflammation (p < .05). Expression levels in patients with heavier fibrosis (stage F4) were higher than in those with less fibrosis (stages F0-3) (p < .01). No gender differences were observed in AR, CCRK, and p-STAT3Tyr705 levels in non-HBV controls; higher levels were observed in HBV-infected males than in HBV-infected females (p < .05). AR, CCRK, and p-STAT3Tyr705 levels in liver tissues positively correlated with each other (p < .0001) and with serum HBV DNA levels (p < .0001). In conclusion, in this study, we first found concordant over-expression of AR, CCRK, and STAT3 in liver tissues of chronic HBV-infected patients who have not yet developed HCC, significantly correlated with the severity of the disease and showed gender differences. STAT3 may be a potential therapeutic co-target for chronic HBV infection.

A booster hepatitis B vaccine for children with maternal HBsAg positivity before 2 years of age could effectively prevent vaccine breakthrough infections.

BACKGROUND: The long-term protective effect of hepatitis B vaccine (HepB), the incidence of hepatitis B virus (HBV) vaccine breakthrough infections (VBIs), and whether a booster HepB is necessary remain to be clarified in children born to mothers with chronic HBV infection. METHODS: Based on a long-term follow-up prospective cohort of 1177 hepatitis B surface antigen (HBsAg)-positive mothers and their paired infants which was established from 2009 to 2011, total 454 children with immunoprophylaxis success as determined by postvaccination serologic testing (PVST) at 7 months old were included in this study. Among the 454 children, 246 never had a booster HepB, and 208 children received a booster HepB from 1 to 5 years of age. Multivariate logistic regression analysis was used to analyse the risk factors for HBV VBIs. RESULTS: The hepatitis B surface antibody (anti-HBs) levels declined sharply from 7 months to 2 years old, and the anti-HBs seronegative rate in the children increased significantly from 2 years old. A total of 31 (6.83%) of the 454 children experienced VBIs, of which 7 had overt and 7 had occult HBV infections. Notably, 14 (45.16%) of the 31 children with VBIs were diagnosed at 2 years old, and all of them had anti-HBs positivity (> 10 mIU/mL) at 1 year old. Maternal hepatitis B e antigen (HBeAg) positivity, higher HBV DNA and HBsAg levels, lower initial infant anti-HBs levels and not receiving a booster HepB were independent risk factors for VBIs. The incidence of VBIs was significantly lower in children with a booster HepB than in nonboosted children (0.50 vs. 11.90%, P < 0.001), and none of the boosted children developed overt or occult HBV infection. The anti-HBs levels of 76.67% for the children with VBIs in the nonboosted group indicated positivity before VBIs was detected. CONCLUSIONS: After the primary full immunization with HepB, children born to mothers with chronic HBV infection, especially the children with maternal HBeAg positivity, high HBV DNA levels, high HBsAg levels and/or low initial infant anti-HBs levels, were at a high risk of VBIs, and a booster HepB for these children before 2 years old, instead of when their anti-HBs level is < 10 mIU/mL, could reduce the incidence of VBIs.

A novel non-invasive score for the prediction of advanced fibrosis in patients with chronic hepatitis B.

INTRODUCTION AND OBJECTIVES: Evaluation of liver fibrosis is important for treatment decisions, complications and to predict prognosis in patients with chronic hepatitis B (CHB). Our aim was to develop a new non-invasive fibrosis scoring method and prove its accuracy in the differentiation of no/low grade and advanced fibrosis in patients with CHB. PATIENTS AND METHODS: Our study included 273 chronic hepatitis B patients who underwent liver biopsy from February, 2007 to February, 2019 with medical records retrospectively reviewed. Preparations of these patients were divided into two groups as ≤ 3 no-low grade fibrosis (n=236) and ≥ 4 advanced fibrosis (n=37) according to histological ISHAK fibrosis scoring system. RESULTS: The newly developed AGAP score and other non-invasive fibrosis scores; Fibrosis-4 index, Aspartate aminotransferase to platelets ratio, Gamma glutamyl transpeptidase to platelet ratio, Goteborg University Cirrhosis Index, King's score, Albumin-bilirubin index, Fibrosis cirrhosis index, Fibrosis index, Fibrosis quotient, Lok score and mean and/or median values of Fibroindex were significantly higher in the advanced fibrosis group compared to the no/low grade fibrosis group (p<0.001). However, there was no significant difference in AAR score among the groups (p=0.265). With cut-off value of 4.038, AUROC value of 0.803, sensitivity of 75.7%, specificity of 73.7% and accuracy of 0.740, AGAP score showed the best performance in advanced fibrosis differentiation compared to 12 other non-invasive fibrosis scoring methods. CONCLUSIONS: The newly developed AGAP score showed better performance in patients with CHB compared to 12 other non-invasive fibrosis scores in differentiation of no/low grade fibrosis and advanced fibrosis.

Molecular characterization of hepatitis B virus (HBV) isolated from a pediatric case of acute lymphoid leukemia, with a delayed response to antiviral treatment: a case report.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) is effectively used as the first-line antiviral for chronic hepatitis B virus (HBV) infection in adults and children older than 12 years. To date, no confirmed case of virologic breakthrough (VBT) in a pediatric case has been reported. CASE PRESENTATION: Here we describe a case of a 5-year old, asymptomatically infected with HBV infection two months after chemotherapy for precursor B acute lymphoblastic leukemia (ALL). Although the 5-year old male is South African, his family originated from Guinea. At the end of the one-year follow-up, the infection progressed to chronic HBV infection, with a high viral load. At 36 weeks (8 months) post-treatment with lamivudine (LAM), there was a partial virologic response (PVR) and after 61 weeks (14 months), he was switched to TDF rescue monotherapy. Even with TDF treatment, he still experienced VBT and subsequent PVR. The full-length genome of HBV isolated 78 weeks after the switch to rescue TDF monotherapy was sequenced and belonged to genotype E. In addition to the LAM mutations (rtS256G and rtM267L), missense mutations in B-cell, T-cell, HLA class I and II-restricted epitopes emerged, which were to evade and escape host surveillance, leading to delayed viral clearance, persistence and disease progression. Two further events of VBT occurred between weeks 113 and 141 of TDF rescue-therapy. Viral loads and liver enzymes are normalizing progressively with long-term therapy. CONCLUSION: Although the host immune reconstitution may be delayed, prolonged TDF treatment was effective in treating this pediatric case of HBV infection with VBT and PVR.

[Ten years of the National Reference Center for hepatitis B viruses and hepatitis D viruses in Giessen, Germany: activities and experiences]. / 10 Jahre Nationales Referenzzentrum für Hepatitis-B-Viren und Hepatitis-D-Viren in Gießen: Tätigkeiten und Erfahrungen.

The National Reference Center (NRC) for hepatitis B viruses (HBV) and hepatitis D viruses (HDV) has been located at the Institute of Medical Virology of the Justus Liebig University (JLU) in Giessen, Germany, since its establishment in 2011. This paper describes the NRC's areas of activity and related experience.The NRC offers comprehensive consulting services on all diagnostic and clinical aspects of acute and chronic HBV and HDV infections for the Public Health Service (ÖGD), diagnostic laboratories, clinics, research institutes, and physicians in private practice. Uncertain diagnostic findings can be analyzed and interpreted and epidemiological correlations clarified with the HBV/HDV special diagnostics established at the NRC using state-of-the-art molecular, biochemical, and genetic laboratory tools. The NRC has access to a strain collection of many well-characterized and cloned HBV/HDV isolates, allowing comparative analysis and evaluation of antiviral resistance mutations and immune escape variants. Together with its national and international partner institutions, the NRC initiates and supervises, among other things, interlaboratory studies for the diagnosis of HBV resistance and immune escape for the establishment and validation of international World Health Organization (WHO) standards and for the improvement of quantitative HDV genome determination. The NRC actively participates in current recommendations and guidelines on HBV and HDV and the recommendations of medical societies. It also highlights current HBV/HDV-relevant aspects with contributions in the form of national and international lectures as well as original articles and comments in national and international journals.

Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B.

BACKGROUND & AIMS: In chronic HBV infection, mitochondrial functions and proteostasis are dysregulated in exhausted HBV-specific CD8 T cells. To better characterise the potential involvement of deregulated protein degradation mechanisms in T cell exhaustion, we analysed lysosome-mediated autophagy in HBV-specific CD8 T cells. Bioactive compounds able to simultaneously target both mitochondrial functions and proteostasis were tested to identify optimal combination strategies to reconstitute efficient antiviral CD8 T cell responses in patients with chronic HBV infection. METHODS: Lysosome-mediated degradation pathways were analysed by flow cytometry in virus-specific CD8 T cells from patients with chronic HBV infection. Mitochondrial function, intracellular proteostasis, and cytokine production were evaluated in HBV-peptide-stimulated T cell cultures, in the presence or absence of the polyphenols resveratrol (RSV) and oleuropein (OLE) and their metabolites, either alone or in combination with other bioactive compounds. RESULTS: HBV-specific CD8 T cells from patients with CHB showed impaired autophagic flux. RSV and OLE elicited a significant improvement in mitochondrial, proteostasis and antiviral functions in CD8 T cells. Cytokine production was also enhanced by synthetic metabolites, which correspond to those generated by RSV and OLE metabolism in vivo, suggesting that these polyphenols may also display an effect after transformation in vivo. Moreover, polyphenolic compounds improved the T cell revitalising effect of mitochondria-targeted antioxidants and of programmed cell death protein 1/programmed cell death ligand 1 blockade. CONCLUSIONS: Simultaneously targeting multiple altered intracellular pathways with the combination of mitochondria-targeted antioxidants and natural polyphenols may represent a promising immune reconstitution strategy for the treatment of chronic HBV infection. LAY SUMMARY: In chronic hepatitis B, antiviral T lymphocytes are deeply impaired, with many altered intracellular functions. In vitro exposure to polyphenols, such as resveratrol and oleuropein, can correct some of the deregulated intracellular pathways and improve antiviral T cell function. This effect can be further strengthened by the association of polyphenols with antioxidant compounds in a significant proportion of patients. Thus, the combination of antioxidants and natural polyphenols represents a promising strategy for chronic hepatitis B therapy.

CD8+ T cells specific to apoptosis-associated epitopes are expanded in patients with chronic HBV infection and fibrosis.

BACKGROUND & AIMS: During chronic viral infections, the apoptosis of activated T cell elicits a CD8+ T cell response directed to those cryptic epitopes that emerge from caspase-cleaved structural proteins. Such response directed to apoptosis-associated epitopes (AEs) contributes to the amplification of immunopathology. METHODS: Here, we have analysed through flow cytometry AE-specific CD8+ T cells in patients with chronic hepatitis B virus (HBV) infection, naïve-to-treatment or undergoing nucleos(t)ide-analogue (NUC) therapy. RESULTS: We found that AE-specific CD8+ T cell frequencies were significantly increased only in those NUC-treated patients who also presented advanced hepatic fibrosis. Regulatory T cells were also expanded in those patients, and AE-specific, but not HBV-specific, CD8+ T cell frequency positively correlated with Treg percentages. Through multiparameter flow cytometry, multidimensionality reduction and unsupervised clustering analysis, we could identify novel subpopulations among effector memory (em) and emCD45RA+ T cell (Tem and Temra) subsets. CD8+ T cells with distinct specificities differentially populated the subpopulation map: while HBV-specific were mostly contained in the Tem subset, AE-specific CD8+ T cells encompassed naïve, as well as T central memory, Tem and Temra cells. CONCLUSION: All together, these findings indicate a link between AE-specific CD8+ T cells and advanced liver fibrosis in patients with chronic HBV infection, and suggest that virus-specific and AE-specific CD8+ T cells exhibit distinct differentiation states and contribute in distinct ways to immunopathology.

Dynamic profile of the HBeAg-anti-HBe system in acute and chronic hepatitis B virus infection: A clinical-laboratory approach.

Wild-type HBV infection is followed by the blood expression of its widely known serological markers of infection, and designated as, hepatitis B virus surface antigen (HBsAg) and its antibody (anti-HBs), anti-HBc antibodies (IgM/IgG), and hepatitis B virus 'e' antigen (HBeAg) and its antibody (anti-HBe). These markers are detected as the infection develops and its kinetic behavior serves as a basis for monitoring the disorder and for diagnosing the clinical form or infection phase. Among these, the HBeAg-anti-HBe system markers demonstrate a dynamic profile whose interpretation, both in the acute or chronic HBV infection context, can offer greater difficulty to the health professionals, due to its particularities. This review offers a revisit to the markers dynamics of this system in the acute and chronic HBV infection and to the clinical and laboratory significance of its expression in these two clinical contexts.

Hepatitis B surface antigen seroclearance: Immune mechanisms, clinical impact, importance for drug development.

HBsAg seroclearance occurs rarely in the natural history of chronic hepatitis B (CHB) infection and is associated with improved clinical outcomes. Many factors are associated with HBsAg seroconversion, including immune and viral factors. However, the immune mechanisms associated with HBsAg seroclearance are still difficult to elucidate. HBsAg seroclearance is the ideal aim of HBV treatment. Unfortunately, this goal is rarely achieved with current treatments. Understanding the mechanisms of HBsAg loss appears to be important for the development of curative HBV treatments. While studies from animal models give insights into the potential immune mechanisms and interactions occurring between the immune system and HBsAg, they do not recapitulate all features of CHB in humans and are subject to variability due to their complexity. In this article, we review recent studies on these immune factors, focusing on their influence on CHB progression and HBsAg seroconversion. These data provide new insights for the development of therapeutic approaches to partially restore the anti-HBV immune response. Targeting HBsAg will ideally relieve the immunosuppressive effects on the immune system and help to restore anti-HBV immune responses.