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Introduction: Patients with resolved hepatitis B virus infection undergoing rituximab are at risk of hepatitis B virus reactivation without antiviral prophylaxis. However, the risk in such patients treated with rituximab-based regimens for membranous nephropathy is not clear. We evaluated the risk of hepatitis B virus reactivation in membranous nephropathy patients with resolved infection undergoing rituximab-based regimens without antiviral prophylaxis., Methods: Clinical data of 51 membranous nephropathy patients with resolved hepatitis B virus infection undergoing rituximab-based regimens without antiviral prophylaxis were retrospectively analyzed. Among these, 21 patients were followed for more than 1 year after rituximab discontinuation. The clinical data collected aimed to assess patients' responses and the risk of hepatitis B virus reactivation during and after rituximab treatment., Results: 30/51 (58.8 %) patients reached complete or partial remission at 12 months. None of the patients experienced HBsAg seroreversion during rituximab treatment. Alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase and total bilirubin levels, as well as the numbers of patients who exceeded the upper limits of normal for alkaline phosphatase and prothrombin time, did not show any statistically significant difference during rituximab-based therapy. Neither did the anti-HBs level, the number of patients with protective anti-HBs titers exceeding 10 U/L, nor the levels of CD19+ B cells, CD4+ T cells, CD8+ T cells, and natural killer cells. Among the 21 patients followed for 12 (ranging from 12 to 19) months after rituximab discontinuation, no hepatitis B virus reactivation was observed. The mean anti-HBs level and the number of patients with anti-HBs titers over 10 U/L did not show any statistically significant difference during the extended follow-up of 33 patient-years. Neither did the CD4+ T cell, CD8+ T cell, nor the natural killer cell counts. One patient presented with an ALT level that exceeded the baseline value by three times and reached above 100 U/L, accompanied by elevations in AST, GGT, and ALP levels. Meanwhile, the anti-HBs titer was 816.09 U/L, and HBsAg was negative., Conclusion: The administration of rituximab-based regimens in membranous nephropathy patients with hepatitis B virus resolved infection leads to a low risk of hepatitis B virus reactivation without antiviral prophylaxis. Patient's immune status, drug combination, rituximab strategy should be fully evaluated when considering antiviral prophylaxis therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Chronic hepatitis B virus (HBV) infection is a serious human health threat given its high morbidity and mortality. Timely and effective antiviral treatment can postpone liver disease progression and reduce the occurrence of HBV-related end-stage liver disease. At present, the antiviral treatment criteria are mainly based on alanine transaminase (ALT) levels, HBV DNA levels and HBV e antigen levels according to the American Association for the Study of Liver Diseases treatment guidelines. However, some chronic hepatitis B (CHB) patients not meeting the above criteria still experience liver disease progression without antiviral treatment. It is urgent to identify a more comprehensive tool to screen out more antiviral treatment candidates as soon as possible., Methods: Considering the vital role of the immune response in the development of HBV infection and CHB cure, we collected data from 335 treatment-naïve CHB patients and comprehensively analysed their clinical and immune traits (including innate and adaptive responses). The immune parameters were obtained by flow cytometry. Finally, we established a model that can better distinguished CHB patients who need treatment through machine learning and LASSO regression of serological and immune parameters., Results: Through a series of analyses, we selected four important clinical parameters (ALT, HBV DNA, the Fibroscan value, and the A/G ratio) and four immune indicators (NKbright + NKp44+, NKbright + NKG2A+, NKT+GranzymeB+, and CD3 + CD107a + ) from more than 200 variables and then successfully established a mathematical model with high sensitivity and specificity to better screen out antiviral treatment candidates from all CHB patients., Conclusions: Our results developed a refined model to better screen out antiviral treatment candidates from all CHB patients by combining common clinical parameters and important immune indicators, including innate and adaptive immunity. These findings provide more information for improving treatment guidelines and have potential implications for the timing of antiviral therapy to achieve better virus control and reduce the occurrence of end-stage liver disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: As nucleos/tide analogue (NA) therapy (e.g. entecavir and tenofovir) for chronic Hepatitis B virus (HBV) infection becomes more widely indicated and available, understanding drug resistance is essential. A systematic review to quantify resistance to these agents has not previously been undertaken., Methods: We performed a systematic review and random-effects meta-analysis to estimate the risk of HBV resistance to entecavir and tenofovir. We searched nine databases up to 29-Aug-23. We included studies of HBV infection featuring >10 individuals, written in English, reporting treatment ≥48 weeks, with assessment of HBV resistance based on viral sequence data. Data were analysed according to prior exposure history to NA, and choice of NA agent. Analyses were performed in R., Findings: 62 studies involving a total of 12,358 participants were included. For entecavir, in treatment-naive individuals (22 studies; 4326 individuals), resistance increased over time to 0.9 % at ≥5 years (95 %CI 0.1-2.3 %), and resistance was increased in NA-experienced individuals (18 studies; 1112 individuals), to 20.1 % (95 %CI 1.6-50.1 %) at ≥5 years. For tenofovir, pooled resistance risk was 0.0 % at all time points, whether previously NA naive (11 studies; 3778 individuals) or experienced (19 studies; 2059 individuals). There was a lack of consistent definitions, poor global representation and insufficient metadata to support subgroup analysis., Interpretation: We have generated the first pooled estimates of HBV entecavir and tenofovir resistance over time. HBV resistance to entecavir in treatment-experienced groups in particular may represent a clinical and public health challenge. To date, tenofovir appears to have an excellent resistance profile, but due to data gaps, we caution that existing studies under-estimate the true real-world risk of resistance. Robust prospective data collection is crucial to reduce health inequities and reduce blind-spots in surveillance as treatment is rolled out more widely., Competing Interests: Declaration of competing interest CC receives doctoral funding support from GSK for a PhD supervised by PCM. YS receives research grant from Gilead Sciences., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Backgrounds & Aims: Given its ability to inhibit HBV replication, Interferon alpha (IFN-α) treatment has been confirmed to be effective in managing Chronic Hepatitis B (CHB). However, its underlying mechanisms are incompletely understood., Methods: Herein, we investigated the antiviral properties of IFN-α by introducing IFN-α expression plasmids into a well-established HBV Hydrodynamic Injection (HDI) mouse model and examined the impact of IFN-α or hepcidin treatment on macrophages derived from THP-1 cells. The cytokine profiles were analyzed using the cytometry microsphere microarray technology, and flow cytometry was used to analyze the polarization of macrophages. Additionally, the IL-6/JAK2/STAT3 signaling pathway and the hepcidin-ferroportin axis were analyzed to better understand the macrophage polarization mechanism., Results: As evidenced by the suppression of HBV replication, injection of an IFN-α expression plasmid and supernatants of IFN-α-treated macrophages exerted anti-HBV effects. The IFN-α treatment up-regulated IL-6 in mice with HBV replication, as well as in IFN-α-treated HepG2 cells and macrophages. Furthermore, JAK2/STAT3 signaling and hepcidin expression was promoted, inducing iron accumulation via the hepcidin-ferroportin axis, which caused the polarization of M1 macrophages. Furthermore, under the effect of IFN-α, IL-6 silencing or blockade downregulated the JAK2/STAT3 signaling pathway and hepcidin, implying that increased hepcidin expression under IFN-α treatment was dependent on the IL-6/JAK2/STAT3 pathway., Conclusion: The IL-6/JAK2/STAT3 signaling pathway is activated by IFN-α which induces hepcidin expression. The resulting iron accumulation then induces the polarization of M1 macrophages via the hepcidin-ferroportin axis, yielding an immune response which exerts antiviral effects against HBV replication., (Copyright © 2024 Elsevier B.V. All rights reserved.)

HBV capsid assembly has been regarded as an attractive potential target for anti-HBV therapy. In this study, we discovery the Novel HBV capsid assembly modulators (CAMs) through structure-based virtual screening and bioassays. A total of 16 structurally diverse compounds were purchased and assayed, including three compounds with inhibition rate > 50% at 20 μM. Further lead optimization based on the most potent compound II-1-7 (EC 50  = 5.6 ± 0.1 µM) were performed by using substructure searching strategy, resulting in compound II-2-9 with an EC 50 value of 1.8 ± 0.6 μM. In bimolecular fluorescence complementation (BiFC) assay, compound II-2-9 inhibited the HBV by disrupting the HBV capsid interactions. In summary, this study provides a highly efficient way to discover novel CAMs, and 2-aryl-4-quinolyl amide derivatives could serve as the starting point for development of novel anti-HBV drugs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

To assess the heterogeneity of HBV reverse transcriptase (RT) quasispecies during 10 years of antiviral therapy and their association with antiviral efficacy. Nineteen patients with chronic hepatitis B (CHB) infection receiving nucleos(t)ide analogues (NAs) were enrolled. Based on the antiviral efficacy after 1 year of treatment, 5 patients were grouped into an early virologic response (EVR) group, while 8 patients were grouped into a late virologic response (LVR) group. Furthermore, 6 CHB patients that had undergone antiviral treatment for 10 years were grouped into a virologic breakthrough (VBT) group. The HBV RT from each patient were amplified, cloned, and sequenced. The complexity of the RT gene in the EVR group was significantly higher than that in the LVR (P = 0.0393) and VBT groups (P = 0.0141). Phylogenetic tree analysis showed that the average branch length of the EVR and LVR groups were significantly greater than that of VBT group (P < 0.001). The complexity (at the nucleotide level) of the RT quasispecies was negatively correlated with the corresponding HBV DNA load (P = 0.0163) at one year post-antiviral treatment. Moreover, both the LVR and VBT groups accumulated more deleterious mutations than the EVR group. After 1 year of NAs treatment, the increased HBV quasispecies complexity and evolutionary topologies, coupled with less deleterious mutations, are likely associated with a favorable efficacy during long-term antiviral treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chronic hepatitis B viral infection is a significant health problem world-wide, and currently available antiviral agents suppress HBV infections, but rarely cure this disease. It is presumed that antiviral agents that target the viral nuclear reservoir of transcriptionally active cccDNA may eliminate HBV infection. Through a series of chemical optimization, we identified a new series of glyoxamide derivatives affecting HBV nucleocapsid formation and cccDNA maintenance at low nanomolar levels. Among all the compounds synthesized, GLP-26 displays a major effect on HBV DNA, HBeAg secretion and cccDNA amplification. In addition, GLP-26 shows a promising pre-clinical profile and long-term effect on viral loads in a humanized mouse model., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: To eliminate mother-to-child transmission (MTCT) of hepatitis B virus (HBV), peripartum antiviral prophylaxis might be required for pregnant women infected with HBV who have a high risk of MTCT despite infant immunoprophylaxis. We aimed to determine the efficacy and safety of peripartum antiviral prophylaxis to inform the 2020 WHO guidelines., Methods: In this systematic review and meta-analysis, we searched PubMed, Embase, Scopus, CENTRAL, CNKI, and Wanfang for randomised controlled trials and non-randomised studies of peripartum antiviral prophylaxis versus placebo or no prophylaxis, with no language restriction, published from database inception until March 28, 2019. We used search terms covering HBV, antiviral therapy, and pregnancy. We included studies that enrolled pregnant women with chronic infection with HBV who received antiviral prophylaxis anytime during pregnancy; that included any of the following antivirals: adefovir, emtricitabine, entecavir, lamivudine, telbivudine, tenofovir alafenamide fumarate, and tenofovir disoproxil fumarate; and that reported the following outcomes: MTCT, indicated by infant HBsAg positivity or HBV DNA positivity, or both, at age 6-12 months, and any infant or maternal adverse events. Two reviewers independently extracted data. Our primary endpoint was MTCT based on infant HBsAg positivity. We assessed pooled odds ratios (ORs) of the efficacy of peripartum antiviral prophylaxis to reduce the risk of MTCT. We assessed safety of prophylaxis by pooling risk differences. The protocol for the systematic review was pre-registered in PROSPERO, CRD42019134614., Findings: Of 7463 articles identified, 595 articles were eligible for full-text review and 129 studies (in 157 articles) were included. The following antivirals were assessed in the meta-analysis: tenofovir disoproxil fumarate 300 mg (19 studies, with 1092 mothers and 1072 infants), lamivudine 100-150 mg (40 studies, with 2080 mothers and 2007 infants), and telbivudine 600 mg (83 studies, with 6036 mothers and 5971 infants). The pooled ORs for randomised controlled trials were similar, at 0·10 (95% CI 0·03-0·35) for tenofovir disoproxil fumarate, 0·16 (0·10-0·26) for lamivudine, and 0·14 (0·09-0·21) for telbivudine. The pooled ORs in non-randomised studies were 0·17 (0·10-0·29) for tenofovir disoproxil fumarate, 0·17 (0·12-0·24) for lamivudine, and 0·09 (0·06-0·12) for telbivudine. We found no increased risk of any infant or maternal safety outcomes after peripartum antiviral prophylaxis., Interpretation: Peripartum antiviral prophylaxis is highly effective at reducing the risk of HBV MTCT. Our findings support the 2020 WHO recommendation of administering antivirals during pregnancy, specifically tenofovir disoproxil fumarate, for the prevention of HBV MTCT., Funding: World Health Organization., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

As a continuation of our previous work, a series of new phenyl acrylamide derivatives (4Aa-g, 4Ba-t, 5 and 6a-c) were designed and synthesized as non-nucleoside anti-HBV agents. Among them, compound 4Bs could potently inhibit HBV DNA replication in wild-type and lamivudine (3TC)/entecavir resistant HBV mutant strains with IC 50 values of 0.19 and 0.18 μM, respectively. Notably, the selective index value of 4Bs was above 526, indicating the favorable safety profile. Interestingly, unlike nucleoside analogue 3TC, 4Bs could significantly inhibit 3.5 kb pgRNA expression. Molecular docking study revealed that 4Bs could fit well into the dimer-dimer interface of HBV core protein by hydrophobic, π-π and H-bond interactions. Considering the potent anti-HBV activity, low toxicity and diverse anti-HBV mechanism from that of nucleoside anti-HBV agent 3TC, compound 4Bs might be a promising lead to develop novel non-nucleoside anti-HBV therapeutic agents, and warranted further investigation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Combinatory enhancement of innate and adaptive immune responses is a promising strategy in immunotherapeutic drug development. Bifunctional macromolecules that simultaneously target two mechanisms may provide additional advantages over the combination of targeting two single pathways. Interferon alpha (IFNα) has been used clinically against viral infection such as the chronic infection of hepatitis B virus (CHB) as well as some types of cancers. OX40 is a costimulatory immune checkpoint molecule involved in the activation of T lymphocytes. To test whether simultaneously activating IFNα and OX40 signaling pathway could produce a synergistic therapeutic effect on CHB and tumors, we designed a bifunctional fusion protein composed of a mouse OX40 agonistic monoclonal antibody (OX86) and a mouse IFNα4, joined by a flexible (GGGGS) 3 linker. This fusion protein, termed OX86-IFN, can activate both IFNα and OX40. We demonstrated that OX86-IFN could effectively activate T lymphocytes in the peripheral blood of mice. Furthermore, we showed that OX86-IFN had superior therapeutic effect to monotherapies in HBV hydrodynamic transfection and syngeneic tumor models. Collectively, our data suggests that simultaneously targeting interferon and OX40 signaling pathways by bifunctional molecule OX86-IFN elicits potent antiviral and antitumor activities, which could provide a new strategy in developing therapeutic agents against viral infection and tumors., (Copyright © 2020. Published by Elsevier B.V.)

Aims: Hepatitis B surface antigen (HBsAg) loss is associated with disease control and improvement of prognosis. Therefore, it is regarded as the optimal treatment endpoint for chronic hepatitis B (CHB) patients. Pegylated interferon (PegIFN)-based extended therapy regimens was assessed in several studies. In order to summarize a conclusion on the HBsAg loss rate and safety in this regimen, a systematic review and meta-analysis was performed., Methods: Studies on Hepatitis B and PegIFN were searched thoroughly in Pubmed, EMBASE, and the Cochrane Library from inception to November 18, 2019. The primary endpoint of this study was the HBsAg loss rate at the end of the extended duration therapy. The secondary endpoint was safety. All analyses were performed by using the R3.6.1 version Software. Quality assessment of RCTs was carried out by using Review manager 5.3., Results: A total of nine studies, including 545 CHB patients met the inclusion criteria. The pooled HBsAg loss rate after PegIFN-based extended duration therapy was 11% (95% CI: 0.05-0.19), I 2  = 82%, P < 0.01(Q test). The extended duration therapy regimen was safe and tolerable. Subgroup analysis showed HBsAg loss rates were 14% (95% CI: 0.04-0.29) and 10% (95% CI: 0.02-0.20) respectively for HBeAg positive and HBeAg negative patients (P = 0.52). HBsAg loss rates were 11%(95%CI:0.03-0.22)and 12%(95%CI:0.04-0.24)respectively for PegIFN monotherapy and PegIFN with Nucleos(t)ide analogs (NAs) therapy (P = 0.84). HBsAg loss rates were 25% (95% CI: 0.19-0.31) and 8% (95% CI: 0.03-0.15) respectively for the advantageous group and non-advantageous group (P = 0.001)., Conclusions: For CHB patients, extended duration of PegIFNα-based treatment for more than 48 weeks is likely to improve HBsAg clearance rate. Specially, the advantageous group will benefit a lot. In addition, the extended duration therapy regimen is safe and tolerable., (Copyright © 2020 Elsevier B.V. All rights reserved.)

HBV infection is a common cause of liver disease with a high burden worldwide. Current therapeutic strategy relies on interferon and nucleos(t)ide-type drugs with the limitation of functional cure. In this study, a structure-based screening of marine natural products from an in-house library was performed to hit HBV inhibitors, and the gorgonian-derived briarane-type diterpenoids showed inhibitory effects against HBV DNA replication in HepAD38 cells. Preliminary analyses of structure-activity relationship demonstrated that a briarane-based scaffold with an 3E,5(16)-diene and a chlorine-substitution at C-6 is required for the anti-HBV activity. Junceellolide B is one of the potent HBV inhibitors exhibiting efficient reduction of HBsAg and HBeAg production in HBV infected HepG2-NTCP cells with a dose-dependent manner (p < 0.001). It also significantly reduced the secreted HBV DNA, HBV RNA, and HBeAg in HepAD38 cells with the EC 50 values of 0.83, 2.87 and 7.75 μM, respectively. Mechanistically, junceellolide B potently inhibited HBV RNA transcription without promoting HBV RNA degradation. RNA-seq analysis indicated that junceellolide B significantly decreased HBV cccDNA-transcripted products accompanying stable down-regulation of the expression of RNA polymerase II related host transcription factors (ZBED6 and ZBTB7B). These findings suggest junceellolide B to be a transcription inhibitor of cccDNA and a promising lead for the development of new anti-HBV agent., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The population of HBV infection with family history of hepatocellular carcinoma (HCC) is the high risk group for the development of HCC. The aim of this study was to evaluate the effect of the de novo combination therapy including pegylated-interferon α-2a (PEG-IFNα-2a) and entecavir (ETV) in this high risk population. The study recruited 58 Hepatitis B e Antigen (HBeAg)-Positive CHB patients patients with HBV-DNA > 10 7  IU/mL, genotype B or C and HCC family history and were treated for 48 weeks. Patients without HBeAg loss at the 48th week were 40 patients and extended the combination therapy to 96 weeks. All patients were followed up to 120 weeks. The rate of HBeAg loss and HBsAg loss was 12/40(30.0%) and 2/40(5.0%) at week 120 respectively. When logistic regression analysis was used to identify viables of HBeAg loss, HBV-DNA levels <20 IU/mL at week 48 was found to have a 6.02 fold increased probability (95% CI = 1.17-30.40, P = .03) of HBeAg loss. Patients with HBV-DNA levels <20 IU/mL at week 48 had a high probability of HBeAg loss 8/17(47.1%), HBsAg loss 2/17(11.8%), compared to 4/23(17.4%), 0/23(0%) in patients with HBV-DNA ≥ 20 IU/mL. Combination therapy for 96 weeks was well tolerated. During the combination therapy, low-level viremia during treatment is reversely associated with response. The combination therapy of PEG-IFNα and ETV was suggested to extend to 96 weeks when HBV-DNA was completed suppressed at week 48., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019. Published by Elsevier B.V.)

Sodium taurocholate cotransporting polypeptide (NTCP) involved in bile acid transport in the liver is an entry receptor of hepatitis B virus (HBV). In the present study, we introduce a mass spectrometric screening assay for targeting HBV entry inhibitors that can reduce NTCP transporter activity by employing taurocholic acid (TCA) labeled with stable isotope (2,2,4,4-d 4 -TCA, d 4 -TCA) and NTCP-overexpressing human liver cancer cell lines such as HepG2 and Huh-7. The accuracy and reliability of the proposed mass spectrometric NTCP activity assay have been validated with known HBV inhibitors including cyclosporine A (CsA) and pre-S1 peptide (PreS/2-48 Myr or myrcludex B analog) that suppress the entry of HBV into hepatocytes by targeting NTCP. For the inhibitor screening assay, NTCP-overexpressing HepG2 or Huh-7 cells are treated with either a combination of TCA and an inhibitor (CsA or PreS/2-48 Myr ) or d 4 -TCA alone to serve as a reference. The activity of an HBV inhibitor is determined by relative quantification between TCA and d 4 -TCA in a 1:1 mixture of inhibitor-treated cells and untreated control cells using liquid chromatography-mass spectrometry. With our new approach, the half maximal inhibitory concentration (IC 50 ) values for CsA and PreS/2-48 Myr have been determined at micromolar and nanomolar concentrations, respectively, which is consistent with the previous results obtained with other conventional HBV entry inhibitor assay methods. Our assay method does not require HBV infection or radioactive 3 H-TCA and provides a facile way to identify viral entry inhibitors via measuring bile acid transport activity of NTCP., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chronic hepatitis B virus (HBV) infection is currently treated with nucleoside/nucleotides analogs. They are potent inhibitors of HBV DNA polymerase, which also functions as reverse transcriptase. Although nucleoside/nucleotide analogs efficiently suppress HBV replication in liver cells, they cannot eradicate HBV DNA from liver cells and cure the disease. Therefore, it is still mandatory to identify and develop effective inhibitors that target a step other than reverse transcription in the viral replication cycle. HBV capsid assembly is a critical step for viral replication and an attractive target for inhibition of HBV replication. We conducted in silico screening of compounds expected to bind to the HBV capsid dimer-dimer interaction site. The selected compounds were further examined for their anti-HBV activity in vitro. Among the test compounds, novel pyrimidotriazine derivatives were found to be selective inhibitors of HBV replication in HepG2.2.15.7 cells. Among the compounds, 2-[(2,3-dichlorophenyl)amino]-4-(4-tert-butylphenyl)-8-methyl-4H,9H-pyrimido[1,2-a][1,3,5]triazin-6-one was the most active against HBV replication. Studies on its mechanism of action revealed that the compound interfered with HBV capsid assembly determined by a cell-free capsid assembly system. Thus, the pyrimidotriazine derivatives are considered to be potential leads for novel HBV capsid assembly inhibitors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The deficiency of nucleos(t)ide analogues (NAs) as anti-hepatitis B virus (HBV) drugs in clinical use is attributable to their insufficient enrichment in liver and non-target organ toxicity. We aimed to develop potent anti-HBV adefovir derivatives with hepatotrophic properties and reduced nephrotoxicity. A series of adefovir mono l-amino acids, mono cholic acid-drug conjugates were designed and synthesized, and their antiviral activity and uptake in rat primary hepatocytes and Na + -dependent taurocholate co-transporting polypeptide (NTCP)-HEK293 cells were evaluated. We isolated compound 6c as the optimal molecular candidate, with the highest antiviral activity (EC 50 0.42 μmol/L, SI 1063.07) and highest cellular uptake in primary hepatocytes and NTCP-HEK293 cells. In-depth mechanistic studies demonstrated that 6c exhibited a lower toxicity in HK-2 cells when compared to adefovir dipivoxil (ADV). This is because 6c cannot be transported by the human renal organic anion transporter 1 (hOAT1). Furthermore, pharmacokinetic characterization and tissue distribution of 6c indicates it has favorable druggability and pharmacokinetic properties. Further docking studies suggested compounds with ursodeoxycholic acid and l-amino acid groups are better at binding to NTCP due to their hydrophilic properties, indicating that 6c is a potential candidate as an anti-HBV therapy and therefore merits further investigation., (Copyright © 2019. Published by Elsevier Ltd.)

Hepatitis B virus (HBV) infection is a major risk factor for chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) worldwide. While multiple hepatitis B drugs have been developed, build up of drug resistance during treatment or weak efficacies observed in some cases have limited their application. Therefore, there is an urgent need to develop substitutional pharmacological agents for HBV-infected individuals. Here, we identified cetylpyridinium chloride (CPC) as a novel inhibitor of HBV. Using computational docking of CPC to core protein, microscale thermophoresis analysis of CPC binding to viral nucleocapsids, and in vitro nucleocapsid formation assays, we found that CPC interacts with dimeric viral nucleocapsid protein (known as core protein or HBcAg) specifically. Compared with other HBV inhibitors, such as benzenesulfonamide (BS) and sulfanilamide (SA), CPC achieved significantly better reduction of HBV particle number in HepG2.2.15 cell line, a derivative of human HCC cells that stably expresses HBV. CPC also inhibited HBV replication in mouse hydrodynamic model system. Taken together, our results show that CPC inhibits capsid assembly and leads to reduced HBV biogenesis. Thus, CPC is an effective pharmacological agent that can reduce HBV particles., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hepatitis B, a viral infectious disease caused by hepatitis B virus (HBV), is a life-threatening disease that leads liver cirrhosis and liver cancer. Because the current treatments for HBV, such as an interferon (IFN) formulation or nucleoside/nucleotide analogues, are not sufficient, the development of a more effective agent for HBV is urgent required. CDM-3008 (1, 2-(2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridin-8-yl)-1,3,4-oxadiazole) (RO8191)) is a small molecule with an imidazo[1,2-a][1,8]naphthyridine scaffold that shows anti-HCV activity with an IFN-like effect. Here, we report that 1 was also effective for HBV, although the solubility and metabolic stability were insufficient for clinical use. Through the structure-activity relationship (SAR), we discovered that CDM-3032 (11, N-(piperidine-4-yl)-2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridine-8-carboxamide hydrochloride) was more soluble than 1 (>30 mg/mL for 11 versus 0.92 mg/mL for 1). In addition, the half-life period of 11 was dramatically improved in both mouse and human hepatic microsomes (T1/2, >120 min versus 58.2 min in mouse, and >120 min versus 34.1 min in human, for 11 and 1, respectively)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chronic hepatitis B virus (HBV) infection remains a serious global health concern. Cisplatin is a chemotherapeutic agent commonly used to treat various cancers. However, HBV-infected patients receiving chemotherapy are at risk of HBV reactivation via unknown mechanisms, which we aimed to elucidate in this study. We found that autophagy plays a central role in cisplatin-induced HBV replication. Cisplatin treatment induced autophagy in both HBV-replicating cells and an HBV-transgenic mouse model as evident from marked upregulation of microtubule-associated protein 1 light chain 3 (LC3)-II and the accumulation of red fluorescent protein (RFP)-LC3 puncta. Cisplatin induced complete autophagic flux, which was detected via monitoring of p62 degradation and RFP-GFP-LC3 expression. Inhibition of autophagy by chloroquine, 3-methyladenine, or Atg5 knockdown significantly attenuated cisplatin-induced HBV replication. Additionally, cisplatin-induced autophagy could be significantly attenuated by using the ROS scavenger N-acetyl-l-cysteine. Mechanically, cisplatin promoted HBV replication and autophagy through ROS/JNK and AKT/mTOR signaling. Inhibition of JNK or activation of Akt/mTOR signaling reversed cisplatin-mediated autophagy and HBV replication promotion. In contrast, suppression of Akt/mTOR signaling further promoted cisplatin-induced HBV replication. Finally, pharmacotherapeutic inhibition of autophagy or ROS production impaired HBV production induced by cisplatin in vivo. Together, our results indicate that ROS/JNK and mTOR/AKT-mediated autophagy plays an important role in cisplatin-induced HBV reactivation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mutation and recombination drive the evolution of most pathogens by generating the genetic variants upon which selection operates. Those variants can, for example, confer resistance to host immune systems and drug therapies or lead to epidemic outbreaks. Given their importance, diverse evolutionary studies have investigated the abundance and consequences of mutation and recombination in pathogen populations. However, some controversies persist regarding the contribution of each evolutionary force to the development of particular phenotypic observations (e.g., drug resistance). In this study, we revise the importance of mutation and recombination in the evolution of pathogens at both intra-host and inter-host levels. We also describe state-of-the-art analytical methodologies to detect and quantify these two evolutionary forces, including biases that are often ignored in evolutionary studies. Finally, we present some of our former studies involving pathogenic taxa where mutation and recombination played crucial roles in the recovery of pathogenic fitness, the generation of interspecific genetic diversity, or the design of centralized vaccines. This review also illustrates several common controversies and pitfalls in the analysis and in the evaluation and interpretation of mutation and recombination outcomes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background and Aims: Much evidence indicates that the soluble antigens secreted by hepatitis B virus (HBV) inhibit the function of the immune system. The aim of this study is to investigate, after treatment with nucleoside (acid) analogs (NAs) and the inhibition of viral replication, whether the immune systems of patients with a peripheral blood HBV-DNA level <1000 IU/mL, hepatitis B e antigen (HBeAg) disappearance, and a decrease in hepatitis B surface antigen (HBsAg) levels could be reconstructed., Methods: The frequency and phenotype of circulating natural killer (NK) cells, dendritic cells (DCs), T-helper (Th) cells, regulatory T (Treg) cells, CD4 + , CD8 + T cells, T follicular helper (Tfh) cells and B cells subtypes were tested by flow cytometry in chronic hepatitis B (CHB) patients and healthy controls (HCs). The levels of HBV-related serum HBsAg, HBeAg, HBV-DNA load, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined., Results: Regarding the innate immune system, an increased frequency of CD56 dim NK cells was found in the therapeutic response (TR) group compared with that in the immune-active phase (IA) group. Additionally, regarding the adaptive immune system, the Th17/CD4 + CD25 + CD127 dim Treg ratio was reduced in the TR group. Additionally, the frequency of CD40L + CXCR5 + CD4 + T cells and CD40 + CD19 + CD27 + CD38 + B cells was significantly higher than that of HCs, while that of PDL1 + CD19 + B cells was lower. Furthermore, the frequencies of CTLA4 + CD4 + T cells and CTLA4 + CD8 + T cells in patients with CHB were significantly higher than those in HCs., Conclusion: After NA treatment and the inhibition of viral replication, circulating CD56 dim NK cells and the balance of Th17/Treg can be recovered. Restoring circulating CD56 dim NK cells and the Th17/Treg balance may help reduce HBsAg levels in patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In lamivudine-refractory chronic hepatitis B (CHB) patients, discontinuation of lamivudine therapy may lead to loss of lamivudine-resistant hepatitis B virus (HBV) and reappearance of wide-type HBV as dominant strains, yet the underlying mechanism remains unclear. In this study, we cloned wide-type and lamivudine-resistant HBV genomes from the sera of a CHB patient who stopped lamivudine therapy after occurrence of resistant virus and determined the biologic properties of the two isolates in hepatoma cell lines. Sequencing reverse transcriptase region of HBV revealed that the patient developed lamivudine-resistant mutations (rtV173 L, rtL180 M, and rtM204 V) 36 months after the start of lamivudine therapy, and lamivudine-resistant mutants reversed to wild-type after the treatment was stopped for 8 months. Our data showed that the wild-type and mutant isolates had similar transcriptional and translational activity. However, comparison of intracellular and released HBV DNA levels showed that replication efficiency of the mutant virus was approximately 50% of wild-type HBV, while the infectivity of released virus was not affected by the lamivudine-resistant mutations. In conclusion, the reversion of lamivudine-resistant mutants to wild-type HBV after discontinuation of lamivudine in hepatitis B patients may be attributed to better replication fitness of wild-type HBV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An estimated 240 million people worldwide are chronically infected with the hepatitis B virus (HBV). Despite readily available vaccination, HBV infections remain highly prevalent. As established HBV infections constitute a strong risk factor for developing hepatocellular carcinoma their treatment is a major task for the health system. Unfortunately, HBV is not curable with today's medicine. Approximately 15 million HBV patients have developed a hepatitis delta (HDV) infection on top of their HBV infection. The patients superinfected with this satellite virus suffer from a more severe disease development. The knowledge of the viruses, their classifications, clinical implications, treatment options and efforts to increase the drug variety are compiled in this review. The current standard therapies include nucleosidic reverse transcriptase inhibitors and interferon. As the known treatments fail to cure HBV and HDV, targeted treatment is highly warranted. The focus of this review is set on the drugs currently under clinical investigation. Furthermore, strategies for the development of targeted treatment, and compounds with novel mode of action are described., (Copyright © 2018. Published by Elsevier B.V.)

Check point inhibitor anti-PD1 antibody produced some efficacy in Hepatocellular Carcinoma (HCC) patients previously treated with sorafenib. Unfortunately, HCC patients with hepatitis B virus (HBV) infection did not respond as well as uninfected patients. Previously, Second mitochondria-derived activator of caspases (SMAC) mimetics-the antagonist for inhibitor of apoptosis proteins (IAPs) can rapidly reduce serum hepatitis B virus DNA in animal model. APG-1387 is a novel SMAC-mimetic, small molecule inhibitor targeting inhibitor of apoptosis proteins (IAPs). In our study, firstly, we found that HCC patients with copy number alteration of cIAP1, cIAP2, and XIAP had a dismal prognosis. Then, we discovered that APG-1387 alone could induce apoptosis of PLC/PRF/5 which was HBV positive both in-vitro and in-vivo. Furthermore, we found that APG-1387 significantly up-regulated the expression of calreticulin and HLA-DR in PLC/PRF/5 via activating non-classic NF-κB pathway. Also, compared to vehicle group, APG-1387 increased NK cell counts by 5 folds in PLC/PRF/5 xenograft model. In-vitro, APG-1387 positively regulated T cells by reducing Treg differentiation and down-regulating PD1 expression in CD4 T cell. Moreover, APG-1387 had no impact on memory T cells. Consequently, our results suggest that APG1387 could be a good candidate to combine with anti-PD1 antibody treatment to overcome low responds of check point inhibitors in HBV positive HCC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: In Australia's Northern Territory, the hepatitis B virus (HBV) subgenotype A2 (subtype adw2) vaccine was introduced in 1988 for Indigenous infants. Subsequently, the circulating viral genotype has been identified as subgenotype C4 (subtype ayw3). We assessed HBV vaccine effectiveness (VE) in light of this subtype mismatch., Methods: Participants of the Aboriginal Birth Cohort (ABC) study were recruited at birth (1987-1990), with HBV serology obtained at follow-up waves 3 (2005-2007) and 4 (2013-2015). Participants were immune if HBV surface antibody levels exceeded 10 IU/L. We determined the VE against any HBV infection (anti-HBc + ) and against chronic infection (HBsAg + or HBV DNA + ), comparing non-vaccinated participants with those fulfilling United States Centers for Disease Control and Prevention (CDC) criteria for full HBV immunisation., Results: Of 686 participants in the ABC study, we obtained HBV serology from 388 at wave 4. 181 participants were immune to HBV and 97 had evidence of any infection. Seven participants were chronically infected, of whom five had received three vaccine doses, and anti-HBc seroconversion had occurred subsequent to the three vaccine doses for two of these seven participants. Comparing the 107 participants who had been vaccinated in accordance with CDC recommendations and 127 who had not been vaccinated, VE against any infection was 67% (95%CI, 43-104%). The odds of being anti-HBc + was 87% lower in participants raised in urban settings compared to those born into families from remote areas (OR, 0.1; 95%CI, 0.03-0.4)., Conclusions: In a setting where there exists a subtype mismatch between vaccine and circulating genotype, the vaccine was largely effective in preventing chronic infection but sub-optimal against any infection. The implications of a high prevalence of anti-HBc seropositivity in this population are unclear and require further study. The fact that anti-HBc seropositivity was strongly associated with remote dwelling suggests ongoing viral exposure in remote settings., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study, a series of 2-arylthio-5-iodo pyrimidine derivatives, as non-nucleoside hepatitis B virus inhibitors, were evaluated and firstly reported as potential anti-HBV agents. To probe the mechanism of active agents, DHBV polymerase was isolated and a non-radioisotopic assay was established for measuring HBV polymerase. The biological results demonstrated that 2-arylthio-5-iodo pyrimidine derivatives targeted HBV polymerase. In addition, pharmacophore models were constructed for future optimization of lead compounds. Further study will be performed for the development of non-nucleoside anti-HBV agents., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hepatitis B virus (HBV) infection is a leading cause of liver damage and hepatic inflammation. Upon infection, effective antiviral responses by CD8+ T cells, CD4+ T cells, Natural killer (NK) cells, and monocytes can lead to partial or complete eradication of the viral infection. To date, many studies have shown that the production of inhibitory cytokines such as Interleukin 10 (IL-10), Transforming growth factor beta (TGF-β), along with dysfunction of the dendritic cells (DCs), and the absence of efficient innate immune responses could lead to T cell exhaustion, development of persistent infection, and inability to eradicate the viral infection from liver. Understanding the immunopathogenesis of the virus could be useful in providing further insights toward novel strategies in the eradication of HBV infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Antiviral therapy has been documented to reduce perinatal transmission of hepatitis B virus (HBV) in highly viremic mothers. This large prospective cohort study conducted in China aims to delineate the maternal viral threshold for consideration of antiviral prophylaxis in settings with limited resources., Methods: A total of 1177 mother-infant pairs with positive maternal hepatitis B surface antigen (HBsAg) under current passive-active prophylaxis regimen were enrolled from community health centers in Jiangsu and Henan provinces, China. Maternal hepatitis B e antigen (HBeAg) status and viral load were tested at 36-40 weeks of gestation. Post-vaccination serologic testing was performed at 7 and 12 months of age., Results: HBeAg-positive mothers (419/1177; 35.6%) had significantly higher viral loads, compared with HBeAg-negative mothers (758/1177; 64.4%) (8.12 vs. 2.69 log IU/mL, p < .0001). Twenty infants, born to HBeAg-positive mothers with high viral loads (median, 8.38; range: 7.82-9.22 log IU/mL), were infected at 7 months of age. In contrast, none of the HBeAg-negative mothers transmitted HBV to their offspring. After adjustment for the other risk factor, a higher maternal viral load was significantly associated with a higher risk of transmission (adjusted odds ratio, 3.78; 95% confidence interval: 1.46-9.81; p = .006). The rates of passive-active immunoprophylaxis failure were 0.0% (0/789), 0.0% (0/27), 0.0% (0/32) and 6.1% (20/329) at maternal viral loads of <5, 5-6, 6-7 and ≥7 log IU/mL, respectively. The antibody to hepatitis B surface antigen (anti-HBs) response rate was 98.4% (1138/1157) at 7 months of age., Conclusions: Results from this study indicate that the maternal viral threshold associated with perinatal transmission of HBV is 7 log IU/mL, which may be appropriate for consideration of antiviral prophylaxis in settings with limited resources., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Hepatitis B virus (HBV) variants are associated with nucleos/tide analogue (NA) response and liver disease but it is unknown whether NA influences extrahepatic HBV persistence., Objectives: To investigate HBV replication and genetic evolution in hepatic and extrahepatic sites of chronic hepatitis B (CHB) before and after NA therapy., Study Design: A total of 13 paired plasma, peripheral blood mononuclear cells (PBMC), were collected from chronic HBV carriers at baseline and after a median 53 weeks NA therapy as well as liver biopsy (N=7 baseline, N=5 follow-up). HBV covalently closed circular DNA (cccDNA) and messenger (m) RNA in liver and PBMC were analyzed. HBV polymerase (P)/surface (S), basal core promoter (BCP)/pre-core (PC)/C gene clonal sequencing was done in plasma, peripheral blood mononuclear cells (PBMC), and liver., Results: Compare to baseline, at ∼53 weeks follow-up, there was no significant change in HBV cccDNA levels in liver (0.2-0.08 copies/hepatocyte, p>0.05) or in PBMC 0.003-0.02 copies/PBMC, p>0.05), and HBV mRNA remained detectable in both sites. At baseline, BCP variants were higher in PBMC vs. liver and plasma. After therapy, drug resistant (DR) and immune escape (IE) variants increased in liver but IE and PC variants were more frequent in PBMC. HBV P/S diversity was significantly higher in PBMC compared to plasma., Conclusion: Continuous HBV replication occurs in liver and PBMC and shows compartmentalized evolution under selective pressure of potent NA therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The hepatitis viruses represent a major public health problem worldwide. Procedures for characterization of the genomic composition of their populations, accurate diagnosis, identification of multiple infections, and information on inhibitor-escape mutants for treatment decisions are needed. Deep sequencing methodologies are extremely useful for these viruses since they replicate as complex and dynamic quasispecies swarms whose complexity and mutant composition are biologically relevant traits. Population complexity is a major challenge for disease prevention and control, but also an opportunity to distinguish among related but phenotypically distinct variants that might anticipate disease progression and treatment outcome. Detailed characterization of mutant spectra should permit choosing better treatment options, given the increasing number of new antiviral inhibitors available. In the present review we briefly summarize our experience on the use of deep sequencing for the management of hepatitis virus infections, particularly for hepatitis B and C viruses, and outline some possible new applications of deep sequencing for these important human pathogens., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: We aimed to study the dynamic changes of hepatitis B virus (HBV) core promoter/precore (CP/preC) sequences during antiviral treatment and their associations with virological responses., Materials and Methods: The baseline and 12-week CP/preC sequences (nts 1655-2014) were obtained from 52 chronic hepatitis B patients with positive hepatitis B e antigen (HBeAg), who received a 104-week lamivudine and adefovir dipivoxil combination therapy. The mutations within the CP/preC were analyzed against genotype specific reference sequences. The nucleotide change rates in individuals during therapy were analyzed in a pairwise comparison manner., Results: There was no significant difference of the mutation rate at each nucleotide site between baseline and week 12 of treatment (P>0.05). The mutation rates of A1762T/G1764A and G1896A were found to decrease from 46.2% (24/52) at baseline to 36.5% (19/52) at week 12 (P=0.426) and from 28.8% (15/52) to 21.2% (11/52) (P=0.497), respectively. The nucleotide change rates varied from 0.0% - 7.8% in individuals [0.0% in Group 1 (N=26); 0.3% - 7.8% in Group 2 (N=26)] during the first 12-week treatment. HBV DNA levels in Group 2 were significantly lower than those in Group 1 throughout therapy (P<0.01) (e.g., 1.5±1.3log 10 IU/ml vs. 2.6±1.0log 10 IU/ml at week 104, P=0.001). At week 104 the rates of HBV DNA undetectable and HBeAg loss in Group 2 were significantly higher than those in Group 1 (P<0.05). Along with the increased nucleotide change rates, the rate of HBV DNA undetectable at week 104 tended to increase (odds ratio=0.323, 95% confidence interval=0.138-0.758, P<0.001)., Conclusion: Our findings suggested that the nucleotide changes within HBV CP/preC region during the first 12-week treatment might be associated with a better virological response., (Copyright © 2017. Published by Elsevier B.V.)

Background: Neutralizing antibodies (anti-HBs) after immunization with hepatitis B virus (HBV) vaccines against HBV surface antigen (HBsAg) wane after 10-15years. We analyzed the effect of an adolescent booster given to vaccination-protected children born to mothers with different HBsAg-carrying status against HBV infection in their mature adulthood., Methods: A total of 9793 individuals, who were HBsAg-negative at childhood (baseline) and donated blood samples, both during childhood and adulthood, from the vaccination group in "Qidong Hepatitis B Intervention Study", were enrolled. Among them 7414 received a one-dose, 10μg-recombinant HBV vaccine booster at 10-14years of age. At endpoint (23-28years of age), we determined the HBV serological markers and quantified their serum HBV-DNA in each of the chronic HBV-infected adults., Results: Fifty-seven adults were identified as chronic HBV infection, indicated by HBsAg(+)&anti-HBc(+) for more than 6months. The individuals who were born to HBsAg-positive mothers (high-risk adults) had significantly increased risk of developing chronic HBV infections in adulthood compared with those who were born to HBsAg-negative mothers; the adjusted odds ratio (OR) was 12.56, 95%CI:7.14-22.08. The seronegative status of anti-HBs at 10-11years of age significantly increased the risk of HBV infections among the high-risk adults. When HBsAg(-)&anti-HBc(+) children who were born to HBsAg-positive mothers 70% of them remained as the status and 10% of them developed HBsAg(+)&anti-HBc(+). While when they were born to HBsAg-negative mothers 1.05% HBsAg(-)&anti-HBc(+) children developed HBsAg(+)&anti-HBc(+) and 24.74% of them remained as the status in 12-18years. One dose of adolescent booster showed significant protection on high-risk adults from chronic HBV infection; P for trend was 0.015., Conclusions: Maternal HBsAg-positive status was an independent risk factor for vaccination-protected children to develop HBV breakthrough infection in adulthood. Adolescent boosters might be appropriate for high-risk individuals who were born to HBsAg-positive mothers when their serum anti-HBs<10mIU/ml., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The US-affiliated Pacific Island countries (USAPI) is an endemic region for hepatitis B virus (HBV) infection. Universal infant hepatitis B vaccination was introduced in the USAPI in the mid-1980s to mitigate the HBV burden. We assessed the impact of universal infant vaccination on the HBV infection prevalence over time among children born in the 1980s, 1990s, and 2000s in the USAPI. Demographic and serologic data from serial sero-surveys conducted between 1985 and 2015 were obtained. Descriptive statistics and analysis of variance were performed. From data obtained from 4827 children (2-11years), HBV prevalence decreased markedly: 8.4% in the 1980s; 2.5% in the 1990s; and 0.2% in the 2000s (P<0.0001) as vaccination coverage increased: 76.4% in the 1980s; 87.3% in the 1990s; and 97.5% in the 2000s (P<0.0001). These findings underscore the protective effect of universal infant hepatitis B vaccination over time on the HBV burden in an HBV endemic region., (Published by Elsevier Ltd.)

Inhibition of hepatitis B virus (HBV) capsid assembly is a novel strategy for the development of chronic hepatitis B (CHB) therapeutics. Herein we described our lead optimization studies including the synthesis, molecular docking studies and structure-activity relationship (SAR) studies of a series of novel heteroaryldihydropyrimidine (HAP) inhibitors of HBV capsid assembly inhibitors, and the discovery of a potent inhibitor of HBV capsid assembly of GLS4 (ethyl 4-[2-bromo-4-fluorophenyl]-6-[morpholino-methyl]-2-[2-thiazolyl]-1,4-dihydro-pyrimidine-5-carboxylate) which is now in clinical phase 2. GLS4 demonstrated potent inhibitory activities in HBV HepG2.2.15 cell assay with an EC 50 value of 1nM, and it also exhibited high potency against various drug-resistant HBV viral strains with EC 50 values in the range of 10-20nM, more potent than the typical HBV polymerase inhibitors such as lamivudine, telbivudine, and entecavir. Pharmacokinetic profiles of GLS4 were favorable and safety evaluation including acute toxicity and repeated toxicity study indicated that GLS4 was safe enough to support clinical experiments in human., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

All patients with advanced chronic kidney disease or on renal replacement therapy should receive active hepatitis B vaccination. The aim of this retrospective cohort study was to investigate the association between the immune response to hepatitis B vaccination and all-cause, cardiovascular or infection-related mortality in incident dialysis patients starting dialysis between 2001 and 2008 (n=426) in two Austrian dialysis centers. Vaccination response was defined as follows: absent anti-HBs antibody titer or a titer <10IU/L was classified as non-response, seroconversion (SC) was defined as a titer ⩾10IU/L, and seroprotection (SP) as a titer ⩾100IU/L. Kaplan-Meier survival curves and multivariable adjusted Cox Proportional Hazards Models were used to determine the association between vaccination response and all-cause, cardiovascular and infection-related mortality. Of all patients 207 (48.6%) were non-responders, SC was observed in 219 (51.4%), SP in 118 (27.7%) patients. During a median follow-up of 51.2 months 228 (53.5%) patients died. Patients with SP and SC showed a significantly lower all-cause (p<0.001 for both) and cardiovascular mortality (p=0.006 for SP, p=0.01 for SC). SP and SC were independently associated with a significant risk reduction for all-cause mortality (SP: HR 0.69, 95% CI 0.49-0.97, p=0.03; SC: HR 0.72, 95% CI 0.55-0.95, p=0.02). In conclusion, achieving seroconversion and seroprotection after active hepatitis B vaccination is associated with significantly reduced all-cause mortality in incident dialysis patients. This simple and readily available tool allows estimation of patient survival independently of other well-known key parameters such as age, gender, the presence of diabetes and markers of malnutrition and inflammation., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Long-term nucleoside analogue (NA) treatment leads to selection for drug-resistant mutations in patients undergoing hepatitis B virus (HBV) therapy. The Abbott RealTime HBV Sequencing assay (Abbott assay; Abbott Molecular Inc., Des Plaines, IL, USA) targets the reverse transcriptase region of the polymerase gene and as such has the ability to detect NA resistance-associated mutations in HBV., Objectives: We evaluated the analytical performance of the Abbott assay and compared its diagnostic performance to that of a laboratory-developed nested-PCR and sequencing method., Study Design: The analytical sensitivity of the Abbott assay was determined using a serially-diluted WHO International Standard. To validate the clinical performances of the Abbott assay and the laboratory-developed assay, 89 clinical plasma samples with various levels of HBV DNA were tested using both assays., Results: The limit of detection of the Abbott assay, was 210IU/ml and it successfully detected mutations when the mutant types were present at levels ≥20%. Among 89 clinical specimens, 43 and 42 were amplification positive in the Abbott and laboratory-developed assays, respectively, with 87.6% overall agreement (78/89; 95% confidence interval [CI], 78.6-93.4). The Abbott assay failed to detect the minor mutant populations in two specimens, and therefore overall concordance was 85.3% (76/89), and the kappa value was 0.79 (95% CI, 0.67-0.90)., Conclusions: The Abbott assay showed comparable diagnostic performance to laboratory-developed nested PCR followed by direct sequencing, and may be useful as a routine method for detecting HBV NA resistance-associated mutations in clinical laboratory settings., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Reports on the concomitant impact of HIV co-infection and long term highly active anti-retroviral therapy (HAART) on the genetic stability and molecular evolution of HBV are limited in sub-Saharan Africa., Materials and Methods: This retrospective study investigated the molecular evolution of chronic HBV in HIV co-infected patients on lamivudine (3TC)-based HAART over a 5year period. Four HIV co-infected patients, consecutively recruited and followed-up, were screened for hepatitis B serological markers, and their viral loads determined. The HBV genome was amplified from longitudinal samples and characterized by Bayesian inference, mutational analysis, and identification of immune selection pressure., Results: All patients exhibited persistent chronic HBV infection at baseline, as well as over the course of follow-up despite exposure to 3TC-based HAART. The polymerase gene in all isolates was relatively variable prior to HAART initiation at baseline and during the course of follow-up, although primary drug resistance mutations were not detected. All but one patient were infected with HBV subgenotype A1. The divergence rates between baseline and the last follow-up sequences ranged from 0 to 2.0×10(-3) substitutions per site per year (s/s/y). Positive selection pressure was evident within the surface and core genes., Conclusion: Despite persistent HBV infection in the HIV co-infected patients exposed to long term 3TC-based HAART, the molecular evolution of HBV over a 5year period was unremarkable. In addition, HBV exhibited minimal genetic variability overtime., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Chronic infection of hepatitis B virus (HBV) is at risk of liver cirrhosis and hepatocellular carcinoma and remains one of the major public health problems worldwide. It is a major barrier of persistence HBV cccDNA under current antiviral therapy as novel strategies of disrupting HBV cccDNA is pressing. The (CRISPR)/Cas9 system is presently emerging in gene editing and we also apply it for targeting and deleting the conserved regions of HBV genome. Two homologous sequences of HBV S and X genes were carried with CRISPR/Cas9 endonuclease to build pCas9 constructs, which may mediate anti-HBV effects of in vitro and in vivo systems in this study. The results showed the better anti-HBV productions by pCas9-2 and without significant differences in between Huh7 and HepG2 cells. CRISPR/Cas9 direct cleavage and mutagenesis were further analyzed of in vitro system. In the M-TgHBV mouse model of HBV, injection of pCas9 constructs by hydrodynamics decreased HBsAg of sera and liver HBcAg. In conclusion, this designed CRISPR/Cas9 system can induce anti-HBV effects and potentially consider as a novel therapeutic agent against chronic HBV infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The matrine-type alkaloid, oxymatrine inhibits hepatitis B virus (HBV) replication but very little is known about these effects in other matrine-type alkaloids, including sophoridine and sophocarpine. Therefore, we compared the in vitro anti-HBV effects of matrine, oxymatrine, sophocarpine, and sophoridine by treating an HBV-transfected cell line (HepG2.2.15) with 0.4-1.6mM of the compounds for 24 or 72h. The levels of the HBV surface antigen (HBsAg) and e antigen (HBeAg) in the culture medium, as well as the intracellular and extracellular HBV DNA levels, were determined. Metabolomic analysis and detection of the mRNA level of p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor receptor-associated factor (TRAF) 6, extracellular signal-regulated kinase (ERK) 1, NOD-like receptor family pyrin domain containing 10 (NLRP10), and caspase-1 were conducted in sophoridine-treated HepG2.2.15 cells. HepG2.2.15 cell exposure to 0.4-1.6mM sophocarpine or sophoridine for 24h reduced the HBsAg level of the medium more effectively than exposure to matrine and oxymatrine did, and reduced the HBeAg levels more effectively than these compounds did at 1.6mM. Sophoridine (0.4-1.6mM) reduced the cell medium HBV DNA levels more than the same concentrations of matrine, oxymatrine, or sophocarpine did. After 72h, 0.4 and 0.8mM sophoridine reduced HBsAg and intracellular HBV DNA levels more potently than matrine, oxymatrine, or sophocarpine did. Furthermore, sophoridine (0.8mM) potently reduced the cell medium HBeAg levels while the metabolomic analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine for 72h exhibited reduced cycloleucine and phytosphingosine levels. In addition, the mRNA expression analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine showed reduced levels of p38 MAPK, TRAF6, ERK1, NLRP10, and caspase-1. Sophoridine produced more potent anti-HBV effects than matrine, oxymatrine, and sophocarpine did. These effects may be related to the sophoridine-mediated reduction of p38 MAPK and TRAF6 levels., (Copyright © 2016. Published by Elsevier B.V.)

Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Chronic hepatitis B virus (HBV) infection (CHB) is a major cause of cirrhosis and hepatocellular carcinoma (HCC). Although the availability of HBV vaccines effectively reduces the incidence of HBV infection, the healthcare burden from CHB remains high. Several antiviral agents, such as (pegylated-) interferon-α and nucleos(t)ide analogs are approved by US FDA for chronic HBV infection management. Entecavir (ETV) and tenofovir disoproxil fumarate (TDF) have been recommended as the first-line anti-HBV drugs for excellent viral suppression with a low risk of antiviral resistance, but the cost and need for essentially life-long treatment are considerable challenges. And none of these current treatments can eradicate the intracellular virus. Given these issues, there is still an unmet medical need for an efficient HBV cure. We summarize here the key developments of antivirals against hepatitis B virus, including HBV replication cycle inhibitors and host immune regulators., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The treatment goal for chronic hepatitis B is true eradication of the hepatitis B virus, but this is rarely achieved with first-line treatment regimens because of an inability to disrupt covalently closed circular DNA and an inadequate host immune response. Therefore, new antiviral agents are needed to target various stages of the hepatitis B virus lifecycle and modulation of the immune system. This Review provides a summary of available regimens with their strengths and limitations, and highlights future therapeutic strategies to target the virus and host immune response. These new agents can hopefully lead to a finite duration of treatment, and provide a functional and durable cure for chronic hepatitis B infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hepatitis B virus (HBV) infection is one of the major causes of chronic liver diseases. The current therapeutics show limited efficacy. In the HBV life cycle, virus core antigen (HBcAg) plays important multiple roles. Blocking the pleiotropic functions of HBcAg may thus represent a promising strategy for anti-HBV replication. In this study, monoclonal antibody (MAb) against core antigen of human HBV was coupled with TAT protein transduction domain (TAT PTD) to form transbody, and the effect on virus replication was evaluated in vitro. The HBV transbody, HBcMAb-TAT PTD conjugate, recognized HBcAg and retained cell-penetrating activity in living cells. In HBV-transfected liver cell line HepG2.2.15, HBV transbody suppressed not only the extracellular HBsAg, HBeAg and HBV DNA, but also the intracellular HBsAg, HBeAg, HBcAg and HBV DNA in a dose-dependent manner. These results indicate that the transbody prepared possesses readily cell-penetrating ability and potent antiviral activity, providing a novel approach, a cell-permeable antibody against HBcAg, for the treatment of HBV infection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A series of novel isosteviol derivatives having C4-amide substituents were synthesized in order to test for antiviral effects against the hepatitis B virus (HBV) in vitro. Among them, IN-4 [N-(propylcarbonyl)-4α-amino-19-nor-ent-16-ketobeyeran] (5) exhibited inhibitory activity against secretion of HBsAg and HBeAg as well as inhibition of HBV DNA replication. Therefore, the mechanism of its antiviral activity was further analyzed using HBV-transfected Huh7 cells. Exposure to IN-4 produced minimal inhibitory effects on viral precore/pregenomic RNA expression. However, expression levels of the 2.4/2.1-kb preS/major S RNA of the viral surface gene significantly decreased, along with intracellular levels of HBV DNA. A promoter activity analysis demonstrated that IN-4 significantly inhibited viral X, S, and preS expression levels but not viral core promoter activities. In particular, IN-4 was observed to significantly inhibit HBV gene regulation by disrupting nuclear factor (NF)-κB-associated promoter activity. In addition, the nuclear expression of p65/p50 NF-κB member proteins was attenuated following IN-4 treatment, while cytoplasmic IκBα protein levels were enhanced. Meanwhile, IN-4 was observed to inhibit the binding activity of NF-κB to putative DNA elements. Furthermore, transfection of a p65 expression plasmid into Huh7 cells significantly reversed the inhibitory effect of IN-4 on HBV DNA levels, providing further evidence of the central role of NF-κB in its antiviral mechanism. It is therefore suggested that IN-4 inhibits HBV by interfering with the NF-κB signaling pathway, resulting in downregulation of viral gene expression and DNA replication., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The synthesis of 9-(β-d-1,3-dioxolan-4-yl)2,6-diaminopurine nucleoside phosphoramidate prodrugs as well as various 2-amino-6-carbamoylpurine dioxolane derivatives and their phosphoramidates prodrugs is reported. Their ability to block HIV and HBV replication along with their cytotoxicity toward HepG2, human lymphocyte, CEM and Vero cells was also assessed., (Published by Elsevier Ltd.)

The compound LPRP-Et-97543 was isolated from Liriope platyphylla roots and was observed to have potential anti-viral effects in HepG2.2.15 cells against hepatitis B virus (HBV). The antiviral mode was further clarified, and the HBV-transfected Huh7 cells were used as the platform. During viral gene expression, LPRP-Et-97543 treatment had apparent effects on the viral precore/pregenomic and S/preS RNA. Promoter activity analysis demonstrated that LPRP-Et-97543 significantly reduced Core, S, and preS but not X promoter activities. Further examination showed that putative signaling pathways were involved in this inhibitory effect, indicating that NF-κB may serve a putative mediator of HBV gene regulation with LPRP-Et-97543. In addition, the nuclear expression of p65/p50 NF-κB member proteins was attenuated with LPRP-Et-97543 and augmented cytoplasmic IκBα protein levels but without affecting the expression of these proteins in HBV non-transfected cells during treatment. Moreover, LPRP-Et-97543 reduced the binding activity of NF-κB protein to CS1 element of HBV surface gene in a gel retardation analysis and inhibited CS1 containing promoter activity in HBV expressed cells. However, HBV transfection significantly enhances CS1 containing promoter activity without compound treatment in cells. Finally, transfection of the p65 expression plasmid significantly reversed the inhibitory effect of LPRP-Et-97543 on the replicated HBV DNA level in HBV positive cells. In conclusion, this study suggests that the mechanism of HBV inhibition by LPRP-Et-97543 may involve the feedback regulation of viral gene expression and viral DNA replication by HBV viral proteins, which interferes with the NF-κB signaling pathway., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background/aims: Current results had demonstrated lamivudine (LAM) contributed to improve liver function and short-term prognosis in patients with hepatitis B virus-related acute-on-chronic liver failure (ACLF), but data concerning the outcome of long-term prognosis are limited. Our objective was to explore the prediction value of early viral response for prognosis and LAM resistance in ACLF patients with lamivudine treatment within 96weeks., Methods: One hundred and forty consecutive subjects were recruited, 76 patients were treated with LAM and supportive treatment (LAM group) and 64 patients only received supportive treatment (non-NAs group). All the patients were followed up until death or 96weeks. The primary end point was overall survival rate at 96weeks, as well as the relationship between the virologic response at weeks 4 or 12 and prognosis and resistance at 96weeks., Results: At 96weeks, the cumulative survival was higher in the LAM group than that in the non-NA group (43/76 (56.58%) vs 9/64 (14.06%), respectively, p=0.000). The survival rate of patients achieved complete viral response (CVR) at week 4 was higher than that of those with partial virologic response (PVR) during the 96-week follow-up (27/29 [93.10%] vs 16/45 [35.56%], p=0.000). In CVR patients, there was a significant improvement in model for end-stage liver failure (MELD) scores compared to PVR. Logistic recurrence indicated that both 4-week CVR and MELD scores were an independent predictor of the 96-week survival. Twelve patients developed LAM resistance (22.22%); all of them came from the PVR at 4weeks., Conclusion: LAM can significantly improve the long-term survival rate, and 4weeks CVR can predict the long-term clinical outcome and LAM-resistant in patients with HBV-related ACLF., (Copyright © 2014 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)