JNJ-64794964 (AL-034/TQ-A3334), a TLR7 agonist, induces sustained anti-HBV activity in AAV/HBV mice via non-cytolytic mechanisms.

JNJ-64794964 (JNJ-4964/AL-034/TQ-A3334), an oral toll-like receptor 7 agonist, is being investigated for the treatment of chronic hepatitis B (CHB), a condition with a high unmet medical need. The anti-hepatitis B (HBV) activity of JNJ-4964 was assessed preclinically in an adeno-associated virus vector expressing HBV (AAV/HBV) mouse model. Mice were treated orally with 2, 6 or 20 mg/kg of JNJ-4964 once-per-week for 12 weeks and then followed up for 4 weeks. At 6 mg/kg, a partial decrease in plasma HBV-DNA and plasma hepatitis B surface antigen (HBsAg) was observed, and anti-HBs antibodies and HBsAg-specific T cells were observed in 1/8 animals. At 20 mg/kg, plasma HBV-DNA and HBsAg levels were undetectable for all animals 3 weeks after start of treatment, with no rebound observed 4 weeks after JNJ-4964 treatment was stopped. High anti-HBs antibody levels were observed until 4 weeks after JNJ-4964 treatment was stopped. In parallel, HBsAg-specific immunoglobulin G-producing B cells and interferon-γ-producing CD4+ T cells were detected in the spleen. In 2/4 animals, liver HBV-DNA and HBV-RNA levels and liver hepatitis B core antigen expression dropped 4 weeks after JNJ-4964 treatment-stop. In these animals, HBsAg-specific CD8+ T cells were detectable. Throughout the study, normal levels of alanine aminotransferase were observed, with no hepatocyte cell death (end of treatment and 4 weeks later) and minimal infiltrations of B and T cells into the liver, suggesting induction of cytokine-mediated, non-cytolytic mechanisms.

Natural killer cells play an important role in virus infection control: Antiviral mechanism, subset expansion and clinical application.

With the global spread of coronavirus disease 2019 (COVID-19), the important role of natural killer (NK) cells in the control of various viral infections attracted more interest, via non-specific activation, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and activating receptors, as well as specific activation, such as memory-like NK generation. In response to different viral infections, NK cells fight viruses in different ways, and different NK subsets proliferate. For instance, cytomegalovirus (CMV) induces NKG2C + CD57 + KIR+ NK cells to expand 3-6 months after hematopoietic stem cell transplantation (HSCT), but human immunodeficiency virus (HIV) induces KIR3DS1+/KIR3DL1 NK cells to expand in the acute phase of infection. However, the similarities and differences among these processes and their molecular mechanisms have not been fully discussed. In this article, we provide a summary and comparison of antiviral mechanisms, unique subset expansion and time periods in peripheral blood and tissues under different conditions of CMV, HIV, Epstein-Barr virus (EBV), COVID-19 and hepatitis B virus (HBV) infections. Accordingly, we also discuss current clinical NK-associated antiviral applications, including cell therapy and NK-related biological agents, and we state the progress and future prospects of NK cell antiviral treatment.

A hepatitis B virus causes chronic infections in equids worldwide.

Preclinical testing of novel therapeutics for chronic hepatitis B (CHB) requires suitable animal models. Equids host homologs of hepatitis C virus (HCV). Because coinfections of hepatitis B virus (HBV) and HCV occur in humans, we screened 2,917 specimens from equids from five continents for HBV. We discovered a distinct HBV species (Equid HBV, EqHBV) in 3.2% of donkeys and zebras by PCR and antibodies against EqHBV in 5.4% of donkeys and zebras. Molecular, histopathological, and biochemical analyses revealed that infection patterns of EqHBV resembled those of HBV in humans, including hepatotropism, moderate liver damage, evolutionary stasis, and potential horizontal virus transmission. Naturally infected donkeys showed chronic infections resembling CHB with high viral loads of up to 2.6 × 109 mean copies per milliliter serum for >6 mo and weak antibody responses. Antibodies against Equid HCV were codetected in 26.5% of donkeys seropositive for EqHBV, corroborating susceptibility to both hepatitis viruses. Deltavirus pseudotypes carrying EqHBV surface proteins were unable to infect human cells via the HBV receptor NTCP (Na+/taurocholate cotransporting polypeptide), suggesting alternative viral entry mechanisms. Both HBV and EqHBV deltavirus pseudotypes infected primary horse hepatocytes in vitro, supporting a broad host range for EqHBV among equids and suggesting that horses might be suitable for EqHBV and HBV infections in vivo. Evolutionary analyses suggested that EqHBV originated in Africa several thousand years ago, commensurate with the domestication of donkeys. In sum, EqHBV naturally infects diverse equids and mimics HBV infection patterns. Equids provide a unique opportunity for preclinical testing of novel therapeutics for CHB and to investigate HBV/HCV interplay upon coinfection.

Microbiota-Meditated Immunity Abnormalities Facilitate Hepatitis B Virus Co-Infection in People Living With HIV: A Review.

Hepatitis B virus (HBV) co-infection is fairly common in people living with HIV (PLWH) and affects millions of people worldwide. Identical transmission routes and HIV-induced immune suppression have been assumed to be the main factors contributing to this phenomenon. Moreover, convergent evidence has shown that people co-infected with HIV and HBV are more likely to have long-term serious medical problems, suffer more from liver-related diseases, and have higher mortality rates, compared to individuals infected exclusively by either HIV or HBV. However, the precise mechanisms underlying the comorbid infection of HIV and HBV have not been fully elucidated. In recent times, the human gastrointestinal microbiome is progressively being recognized as playing a pivotal role in modulating immune function, and is likely to also contribute significantly to critical processes involving systemic inflammation. Both antiretroviral therapy (ART)-naïve HIV-infected subjects and ART-treated individuals are now known to be characterized by having gut microbiomic dysbiosis, which is associated with a damaged intestinal barrier, impaired mucosal immunological functioning, increased microbial translocation, and long-term immune activation. Altered microbiota-related products in PLWH, such as lipopolysaccharide (LPS) and short-chain fatty acids (SCFA), have been associated with the development of leaky gut syndrome, favoring microbial translocation, which in turn has been associated with a chronically activated underlying host immune response and hence the facilitated pathogenesis of HBV infection. Herein, we critically review the interplay among gut microbiota, immunity, and HIV and HBV infection, thus laying down the groundwork with respect to the future development of effective strategies to efficiently restore normally diversified gut microbiota in PLWH with a dysregulated gut microbiome, and thus potentially reduce the prevalence of HBV infection in this population.

Quantification, epitope mapping and genotype cross-reactivity of hepatitis B preS-specific antibodies in subjects vaccinated with different dosage regimens of BM32.

BACKGROUND: Chronic hepatitis B virus (HBV) infections are a global health problem. There is a need for therapeutic strategies blocking continuous infection of liver cells. The grass pollen allergy vaccine BM32 containing the preS domain of the large HBV surface protein (LHBs) as immunogenic carrier induced IgG antibodies in human subjects inhibiting HBV infection in vitro. Aim of this study was the quantification, epitope mapping and investigation of HBV genotype cross-reactivity of preS-specific antibodies in subjects treated with different dosage regimens of BM32 METHODS: Hundred twenty eight grass pollen allergic patients received in a double-blind, placebo-controlled trial five monthly injections of placebo (aluminum hydroxide, n= 34) or different courses of BM32 (2 placebo + 3 BM32, n= 33; 1 placebo + 4 BM32, n= 30; 5 BM32, n= 31). Recombinant Escherichia coli-expressed preS was purified. Overlapping peptides spanning preS and the receptor-binding sites from consensus sequences of genotypes A-H were synthesized and purified. Isotype (IgM, IgG, IgA, IgE) and IgG subclass (IgG1-IgG4) responses to preS and peptides were determined by ELISA at baseline, one and four months after the last injection. IgG1 and IgG4 subclass concentrations specific for preS and the receptor-binding site were measured by quantitative ELISA. FINDINGS: Five monthly injections induced the highest levels of preS-specific IgG consisting mainly of IgG1 and IgG4, with a sum of median preS-specific IgG1 and IgG4 concentrations of >135 µg/ml reaching up to 1.8 mg/ml. More than 20% of preS-specific IgG was directed against the receptor-binding site. BM32-induced IgG cross-reacted with the receptor-binding domains from all eight HBV genotypes A-H. INTERPRETATION: BM32 induces high levels of IgG1 and IgG4 antibodies against the receptor binding sites of all eight HBV genotypes and hence might be suitable for therapeutic HBV vaccination. FUNDING: This study was supported by the PhD program IAI (KPW01212FW), by Viravaxx AG and by the Danube-ARC funded by the Government of Lower Austria. Rudolf Valenta is a recipient of a Megagrant of the Government of the Russian Federation, grant No 14.W03.31.0024.

Structure guided maturation of a novel humanized anti-HBV antibody and its preclinical development.

We have reported that E6F6, a mouse monoclonal antibody, is a promising treatment option for patients with chronic hepatitis B (CHB). A humanized E6F6 antibody B11 with affinity loss was obtained by CDR-grafting approach. To address this issue, in silico affinity maturation through scanning mutagenesis using CHARMM force field methods was performed on an predicted immune complex model of the B11:HBsAg. We chose four variants with top increased interaction energy for further characterization. The antibody huE6F6-1 within two point mutations (Heavy Chain: Asp65Val; His66Leu) was identified to restore the parental antibody's high binding affinity, neutralization activity, and potent efficacy of viral suppression in vivo. Crystal structure (1.8 Å resolution) based molecular docking proved more stabilized and compact hydrogen bond interactions formed in huE6F6-1.The smaller and dispersed HBV immune complexes of huE6F6-1 by electron microscopy suggested it will have the same therapeutic efficacy as the parental E6F6 mAb. Preclinical study and pharmacokinetics of huE6F6-1 demonstrated that it is a stable and desirable lead candidate to improve the clinical management of CHB. Notably, our structure guided approach may facilitate the humanization and affinity maturation of other rodent antibody candidates during drug development.

Increased Coffee Intake Reduces Circulating HBV DNA and HBsAg Levels in HBeAg-Negative Infection: A Cohort Study.

Coffee is hepatoprotective and potentially antiviral; however, its anti-hepatitis B virus (anti-HBV) property is not known in humans. This study investigated the influence of coffee drinking behaviour as well as clinical and biochemical profiles of hepatitis B e antigen (HBeAg) negative participants on circulating HBV DNA and hepatitis B surface antigen (HBsAg) levels at a 24-week interval. Exactly 114 chronically HBV-infected adult participants were enrolled from the University of Malaya Medical Centre (UMMC), Malaysia. A significant reduction of HBV DNA level was observed in those drinking three or more cups of coffee per day, with a median reduction of 523 IU/mL (P = 0.003). Reduction of HBsAg level was observed in those drinking two cups per day, with a median reduction of 37 IU/mL (P < 0.001). Multivariate analysis showed that increased coffee intake (P = 0.015) and lower ALT level (P = 0.033) were the significant predictors for a lower HBV DNA level, whereas increased coffee intake (P = 0.002) and having a family history of HBV infection (P = 0.021) were the significant predictors for a lower HBsAg level. These data suggest that drinking three cups or more coffee per day reduces circulating HBV DNA and HBsAg levels.

Nivolumab in advanced hepatocellular carcinoma: Sorafenib-experienced Asian cohort analysis.

BACKGROUND & AIMS: Nivolumab, an immune checkpoint inhibitor, is approved in several countries to treat sorafenib-experienced patients with HCC, based on results from the CheckMate 040 study (NCT01658878). Marked differences exist in HCC clinical presentation, aetiology, treatment patterns and outcomes across regions. This analysis assessed the safety and efficacy of nivolumab in the Asian cohort of CheckMate 040. METHODS: CheckMate 040 is an international, multicentre, open-label, phase I/II study of nivolumab in adults with advanced HCC, regardless of aetiology, not amenable to curative resection or local treatment and with/without previous sorafenib treatment. This analysis included all sorafenib-experienced patients in the intent-to-treat (ITT) overall population and Asian cohort. The analysis cut-off date was March 2018. RESULTS: There were 182 and 85 patients in the ITT population and Asian cohort, respectively. In both populations, most patients were older than 60 years, had BCLC (Barcelona Clinic Liver Cancer) Stage C disease, and had received previous systemic therapy. A higher percentage of Asian patients had HBV infections, extrahepatic metastases and prior therapies. Median follow-up was 31.6 and 31.3 months for the ITT and Asian patients, respectively. Objective response rates were 14% and 15% in the ITT population and Asian cohort, respectively. In the Asian cohort, patients with HBV, HCV or those who were uninfected had objective response rates of 13%, 14% and 21%, respectively. The median duration of response was longer in the ITT (19.4 months) vs. Asian patients (9.7 months). Median overall survival was similar between the ITT (15.1 months) and Asian patients (14.9 months), and unaffected by aetiology in Asian patients. The nivolumab safety profile was similar and manageable across both populations. CONCLUSION: Nivolumab safety and efficacy are comparable between sorafenib-experienced ITT and Asian patients. LAY SUMMARY: The CheckMate 040 study evaluated the safety and efficacy of nivolumab in patients with advanced hepatocellular carcinoma who were refractory to previous sorafenib treatment or chemotherapy. This subanalysis of the data showed that treatment responses and safety in patients in Asia were similar to those of the overall treatment population, providing support for nivolumab as a treatment option for these patients. Clinical trial number: NCT01658878.

Hepatitis B seroprevalence in 10-25-year-olds in Mexico - the 2012 national health and nutrition survey (ENSANUT) results.

OBJECTIVES: To estimate hepatitis B virus (HBV) seroprevalence from natural infection or vaccination in 10-25-year-olds in Mexico, using the 2012 National Health and Nutrition Survey (ENSANUT). METHODS: Randomly selected serum samples (1,581) from adolescents and young adults, representative of 38,924,584 Mexicans, were analyzed to detect hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs) and hepatitis B core antibody (anti-HBc). Weighted HBV seroprevalence in the Mexican population and association with sociodemographic variables were calculated. RESULTS: Overall weighted seroprevalence from natural infection (positive for anti-HBs and anti-HBc) was 0.23% (95% confidence interval [95% CI] 0.10-0.52). No HBsAg was detected, indicating no acute or chronic infection. Vaccine-derived immunity (positive ≥ 10.0 mIU/ml for anti-HBs and negative to anti-HBc) was 44.7% (95% CI: 40.2-49.4) overall; lower in persons aged 20-25 years (40.83%) than in persons aged 10-19 years (47.7%). Among the population analyzed, 54.2% (95% CI: 49.6-58.8) were seronegative to HBV (negative for all three markers) and no sociodemographic risk factors were identified. CONCLUSIONS: HBV seroprevalence from natural infection was low. Vaccination-induced immunity was higher among Mexican adolescents than young adults, possibly due to vaccination policies since 1999.

Anti-hepatitis B virus effects of the traditional Chinese herb Artemisia capillaris and its active enynes.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia capillaris (Yin-Chen) is a famous traditional Chinese medicine (TCM) for treating acute and chronic hepatitis in China. Enynes are one type of characteristic constituents in this herb, while their anti-hepatitis B virus (anti-HBV) properties have not been systemically investigated. AIM OF THE STUDY: This study is to reveal the active part of A. capillaris, and systemically investigate the enynes and their anti-HBV activity. MATERIALS AND METHODS: The total extract and each fraction of A. capillaris were assayed for the anti-HBV activity to reveal the active part. Bioassay-guided fractionation using various chromatographic techniques yielded the enynes, whose structures were elucidated by spectroscopic analyses and ECD calculations. The anti-HBV properties inhibiting HBsAg and HBeAg secretions and HBV DNA replication were evaluated on HepG 2.2.15 cell line in vitro. RESULTS: ACT-2 and ACT-3 was revealed to be the respective active and toxic part of A. capillaris. Twelve enynes (1-12) involving four new ones (1-4) and two unusual enyne analogs (13-14) were isolated from the active part (ACT-2). All the isolates were assayed for their anti-HBV activity, and the preliminary structure-activity relationships were summarized based on the structural features. In particular, compound 4 could significantly inhibit the secretions of HBsAg and HBeAg, and HBV DNA replication with IC50 values of 197.2 (SI > 5.1), 48.7 (SI > 20.5) and 9.8 (SI > 102) µM. CONCLUSIONS: Enynes are responsible for the anti-HBV effects of A. capillaris. Hydroxyl and glycosyl groups are preferable for maintaining activity. This is the first time to systematically investigate the anti-HBV activity of enynes in A. capillaris, which provides valuable information for understanding the ethnopharmacological application of Yin-Chen.

Modulators of innate immunity as novel therapeutics for treatment of chronic hepatitis B.

The first line defense mechanisms against viral infection are mediated by the innate immune system. Viral components are detected by infected cells and/or innate immune cells that express different sensory receptors. They in turn mediate induction of direct antiviral mechanisms and further modulation of innate and adaptive immune responses. For evading the innate system, most viruses have evolved efficient mechanisms to block sensing and/or antiviral functions of the innate response. Interestingly, hepatitis B virus (HBV) seems to act like a stealth virus that escapes cell intrinsic antiviral mechanisms through avoiding recognition by the innate system rather than blocking its effector functions. In line with this concept, agonistic activation of innate immunity has emerged as a promising novel anti-HBV therapy approach with several compounds having advanced to the clinical stage.

HBVcircle: A novel tool to investigate hepatitis B virus covalently closed circular DNA.

BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as a stable episome in infected hepatocytes and serves as a template for the transcription of all viral genes. Due to the narrow host range of HBV, the development of a robust mouse model that supports cccDNA-dependent viral replication is a key hurdle in the development of novel HBV therapeutics. This study aimed to develop a novel tool to investigate HBV cccDNA. METHODS: Through minicircle technology, HBVcircle, a recombinant cccDNA, was easily generated and extracted from a genetically engineered E. coli strain. We characterized the performance of HBVcircle in cell culture by transfection and in immunocompetent mice by hydrodynamic injection (HDI). RESULTS: We demonstrated that HBVcircle formed authentic cccDNA-like molecules in vitro in transiently transfected hepatic cells and in vivo in mouse liver after HDI. HBVcircle supported high levels and persistent HBV replication. In addition, we investigated different factors affecting HBV in vivo replication and persistence, including the host genetic background, vector design and dosage, viral genes and genotypes, and immune activation status. Furthermore, different classes of anti-HBV drugs were also assessed with the HBVcircle system. CONCLUSION: Compared with previous reported HBV mouse models which employ other viral vectors to introduce overlength HBV genomes, viral gene expression and associated phenotypes are entirely driven by cccDNA-like viral genomes in the HBVcircle mouse model. Therefore, the HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery. LAY SUMMARY: To establish a mouse model that supports cccDNA-dependent transcription, a novel tool named HBVcircle, was developed with minicircle technology. HBVcircle formed authentic cccDNA-like molecules in hepatocytes, and supported high levels and persistent HBV replication in vivo. The HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.

Immunotherapy With the PreS-based Grass Pollen Allergy Vaccine BM32 Induces Antibody Responses Protecting Against Hepatitis B Infection.

BACKGROUND: We have constructed and clinically evaluated a hypoallergenic vaccine for grass pollen allergy, BM32, which is based on fusion proteins consisting of peptides from the IgE binding sites of the major grass pollen allergens fused to preS (preS1+preS2), a domain of the hepatitis B virus (HBV) large envelope protein which mediates the viral attachment and entry. Aim of this study was the characterization of the HBV-specific immune response induced by vaccination of allergic patients with BM32 and the investigation of the vaccines' potential to protect against infection with HBV. METHODS: Hepatitis B-specific antibody and T cell responses of patients vaccinated with BM32 were studied using recombinant preS and synthetic overlapping peptides spanning the preS sequence. The specificities of the antibody responses were compared with those of patients with chronic HBV infection. Furthermore, the capacity of BM32-induced antibodies, to inhibit HBV infection was investigated using HepG2-hNTCP cell-based in vitro virus neutralization assays. FINDINGS: IgG antibodies from BM32-vaccinated but not of HBV-infected individuals recognized the sequence motif implicated in NTCP (sodium-taurocholate co-transporting polypeptide)-receptor interaction of the hepatitis B virus and inhibited HBV infection. INTERPRETATION: Our study demonstrates that the recombinant hypoallergenic grass pollen allergy vaccine BM32 induces hepatitis B-specific immune responses which protect against hepatitis B virus infection in vitro.

Controversy and Strategies Exploration in Blocking Mother-to-Child Transmission of Hepatitis B.

Hepatitis B, a serious infectious disease caused by the hepatitis B virus (HBV), remains a worldwide social and public health problem. Hepatitis B has a particularly high incidence rate in the world, whereas approximately 35-50% HBV carriers are infected through vertical transmission. Even after newborn immunoprophylaxis, vertical transmission still accounts for 5-10% in China according to plenty of literature in Chinese language. For these reasons, it is important to determine how to effectively intervene in mother-to-child transmission (MTCT). To date, though, intervention methods and measures remain controversial. In order to understand the mechanism of MTCT intervention further and develop effective preventions and interventions, a comprehensive analysis and presentation on some of its more controversial issues will be given in this paper. And eventually we conclude three measures and strategies for these issues: (1) emancipate the mind and seek truth from facts to understand the controversial issues pertaining to MTCT of HBV; (2) treat the basic rules and changing characteristics of MTCT blocking process of hepatitis B with holistic medical thought dialectically and (3) further explore the interaction of genetic susceptibility and environmental factors of MTCT of hepatitis B.

Mutations in pre-core and basic core promoter regions of hepatitis B virus in chronic hepatitis B patients.

AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related indexes. METHODS: One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCR-reverse dot blot hybridization and mismatch amplification mutation assay (MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed. RESULTS: Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen (HBeAg) positive patients, and 84.6% and 96.2%, respectively, in HBeAg negative patients. The rate of pre-C mutations was significantly higher in HBeAg negative patients than in HBeAg positive patients (χ (2) = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients (χ (2) = 2.43, P = 0.12). The presence of mutations in the pre-C (Wilcoxon W = 1802.5, P = 0.00) and BCP regions (Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci (P < 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively. CONCLUSION: HBeAg negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function.

Alternative Therapies for Chronic Hepatitis B Patients With Partial Virological Response to Standard Entecavir Monotherapy.

BACKGROUND: Entecavir (ETV) is a first-line, oral antinucleoside agent for the treatment of chronic hepatitis B patients. Despite its high potency, some patients may still be viremic after prolonged therapy with ETV monotherapy. Long-term outcome data comparing maintained ETV monotherapy to alternative therapies in persistently viremic patients are limited. Our goal was to compare complete viral suppression (CVS) rates [hepatitis B DNA (HBV DNA)<40 to 60 IU/mL] with alternative therapies to continued ETV monotherapy in ETV partial responders. METHODS: This is a retrospective cohort study consisting of 86 consecutive treatment-naive, ETV=0.5 mg partial responders (detectable HBV DNA after ≥12 mo on ETV) who maintained ETV=0.5 mg daily (n=29) or switched to either ETV=1.0 mg daily (n=32) or ETV/tenofovir (TDF)=0.5 mg/300 mg (n=25) in 3 US GI/liver clinics from January 2005 to January 2012. Patients were identified by International Classification of Diseases, Ninth Revision query and data were collected by individual chart review. For those who remained on ETV=0.5 mg, comparison at regimen "switch time" was done using values at 12 months from initial ETV therapy. Rates of CVS were evaluated using Kaplan-Meier methods. Multivariate Cox proportional hazard models were used to estimate hazard ratio (HR) relating to potential predictors to the desirable outcomes of CVS. RESULTS: In all therapy groups, the majority of patients were Asian (93.1% to 100.0%), male (64.0% to 68.8%), and hepatitis B e antigen-positive (95.8% to 100.0%) and had similar baseline alanine aminotransferase (ALT) levels. However, baseline HBV DNA (7.0 vs. 7.9 vs. 7.8 log10 IU/mL, P=0.05) and HBV DNA at regimen switch point (2.9 vs. 3.7 vs. 3.6 log10 IU/mL, P=0.0014) were lower in the ETV=0.5 mg cohort compared with those switched to ETV=1.0 mg or ETV/TDF, respectively. The ETV=0.5 mg cohort also had the shortest duration of ETV=0.5 mg therapy before switch (11.8 vs. 13.5 vs. 19.2 mo, P<0.0001). After the switch point, more patients on ETV/TDF achieved CVS compared with those on ETV=0.5 mg or ETV=1.0 mg at month 6 (77.3% vs. 13.8% vs. 9.4%), month 12 (86.4% vs. 40.5% vs. 25.0%), and month 18 (100% vs. 70.2% vs. 33.3%). Compared with the ETV=0.5 mg and ETV=1.0 mg groups, the ETV/TDF group also had higher rates of ALT normalization at month 6 (73.0% vs, 46.4% vs. 63.0%), month 12 (79.7% vs. 69.5% vs. 77.9%), and month 18 (100.0% vs. 69.5% vs. 86.8%), respectively. The multivariate analyses, inclusive of baseline age and treatment duration on initial therapy with ETV=0.5 mg, indicated that the ETV/TDF combination (HR=12.19, P<0.0001) was independently and positively associated with CVS, whereas high HBV DNA levels at baseline (HR=0.77, P=0.02) and at switch point (HR=0.46, P=0.002) were negatively associated with CVS. ETV=1.0 mg dose was not a predictor for CVS compared with ETV=0.5 mg. CONCLUSIONS: Following adjustments for HBV DNA levels and prior treatment duration, ETV/TDF combination therapy independently predicted superior viral suppression and ALT normalization in partial responders to ETV=0.5 mg daily compared with ETV=0.5 mg or ETV=1.0 mg monotherapy. In patients who continued to be viremic after 12 months of ETV=0.5 mg, one third were still viremic after another 18 months on the same therapy. Alternative therapies should be considered for these patients.

AIC649 Induces a Bi-Phasic Treatment Response in the Woodchuck Model of Chronic Hepatitis B.

AIC649 has been shown to directly address the antigen presenting cell arm of the host immune defense leading to a regulated cytokine release and activation of T cell responses. In the present study we analyzed the antiviral efficacy of AIC649 as well as its potential to induce functional cure in animal models for chronic hepatitis B. Hepatitis B virus transgenic mice and chronically woodchuck hepatitis virus (WHV) infected woodchucks were treated with AIC649, respectively. In the mouse system AIC649 decreased the hepatitis B virus titer as effective as the "gold standard", Tenofovir. Interestingly, AIC649-treated chronically WHV infected woodchucks displayed a bi-phasic pattern of response: The marker for functional cure--hepatitis surface antigen--first increased but subsequently decreased even after cessation of treatment to significantly reduced levels. We hypothesize that the observed bi-phasic response pattern to AIC649 treatment reflects a physiologically "concerted", reconstituted immune response against WHV and therefore may indicate a potential for inducing functional cure in HBV-infected patients.

Current status of immunomodulatory therapy in chronic hepatitis B, fifty years after discovery of the virus: Search for the "magic bullet" to kill cccDNA.

Chronic hepatitis B (CHB) is currently treated with IFN-α and nucleos(t)ide analogues, which have many clinical benefits, but there is no ultimate cure. The major problem consists in the persistence of cccDNA in infected hepatocytes. Because no antiviral drug has been evaluated which significantly reduces copies of cccDNA, cytolytic and noncytolytic approaches are needed. Effective virus-specific T- and B-cell responses remain crucial in eliminating cccDNA-carrying hepatocytes and for the long-term control of HBV infection. Reduction of viremia by antiviral drugs provides a window for reconstitution of an HBV-specific immune response. Preclinical studies in mice and woodchucks have shown that immunostimulatory strategies, such as prime-boost vaccination and PD-1 blockade, can boost a weak virus-specific T cell response and lead to effective control of HBV infection. Based on data obtained in our preclinical studies, the combination of antiviral drugs and immunomodulators may control HBV viremia during a patient's drug-off period. In this article, we review current immune-modulatory approaches for the treatment of chronic hepatitis B and the elimination of cccDNA in preclinical models. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis".

Three new anti-HBV active constituents from the traditional Chinese herb of Yin-Chen (Artemisia scoparia).

ETHNOPHARMACOLOGICAL RELEVANCE: Yin-Chen is a famous traditional Chinese medicine (TCM) in China for the treatment of acute and chronic hepatitis. Two species, namely Artemisia scoparia and Artemisia capillaris, are documented in Chinese Pharmacopoeia as the authentic resources for Yin-Chen. Previous investigation has proved that chlorogenic acid analogs and phenolic acids are two main types of the anti-HBV active constituents of A. capillaris. However, there is no investigation concerned with the anti-HBV components of A. scoparia. AIM OF THE STUDY: The aim of the present study is to recognize the new anti-HBV constituents of A. scoparia by detailed LCMS analyses. MATERIALS AND METHODS: LCMS and bioassay-guided fractionation on the active part of A. scoparia led to the isolation of three new compounds. Their structures were determined by detailed spectroscopic analyses. Anti-HBV assay involving inhibition on HBsAg and HBeAg secretions and HBV DNA replication were performed in virto on HepG 2.2.15 cell line. RESULTS: The 90% ethanol extract of A. scoparia was revealed with anti-HBV activity for the first time, which was further separated into several fractions by column chromatography. Fr. D-4 was revealed with the highest anti-HBV activity, from which three new compounds including one unusual 4-pyridone glucoside (1) and two polyacetylene glucosides (2-3) were isolated under the guidance of LCMS analyses. Compounds 1-3 exhibited activity against the secretions of HBsAg and HBeAg, and HBV DNA replication. In particular, compounds 2 and 3 inhibited HBV DNA replication with IC50 values of 0.07 ± 0.04 and 0.012 ± 0.05 mM, with SI values of 23.6 and 17.1, respectively. Based on the MS/MS experiment, the fragmentation pathways of 1 in both positive and negative modes, and 2 and 3 in negative mode were proposed. The ion pairs of 388-208 (positive) and 432-206 (negative) for 1, 503-341 (negative) for 2, and 503-203 (negative) for 3, could be recognized as their respective diagnostic ions. CONCLUSIONS: The first time investigation on the anti-HBV constituents of A. scoparia yielded three new active compounds, which will provide valuable information for understanding the ethnopharmacological usage of Yin-Chen, as well as the chemical difference with A. capillaris.

Effect of Radix Trichosanthis and Trichosanthin on Hepatitis B Virus in HepG2.2.15 Cells.

Radix Trichosanthis is a Chinese herbal medicine that has great medical value and pharmacological actions. There is already a long history of using the plant Radix Trichosanthis as treatment for hepatitis B virus in China. This research mainly focused on investigating the therapeutic effect of different extracts from Radix Trichosanthis on hepatitis B virus, on a cellular level (ex vivo). Cell survival rate of HepG2.2.15 cells was detected by MTT assay. HBsAg and HBeAg in HepG 2.2.15 cell supernatant were evaluated by enzyme linked immunosorbent assay (ELISA). Results showed that water extract from Radix Trichosanthis had a stronger inhibitive effect on expression of HBsAg and HBeAg in HepG2.2.15 cells than the alcohol extract from the same plant. Considering that the most active component of Radix Trichosanthis was in its aqueous extract and this might be related to the active component Trichosanthin. Trichosanthin was further used for related experiments to confirm this hypothesis. The results showed that Trichosanthin, in the aqueous extract from Radix Trichosanthis, is likely the main component responsible for the anti-hepatitis B viral effect.