Molecular epidemiology of hepatitis B among Indigenous Australians in Queensland and the Torres Strait Islands.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is a major health problem for all Indigenous Australians. Post-2000, Hepatitis B surface antigen prevalence has decreased, although remaining four times higher among Indigenous compared with non-Indigenous people. AIMS: This study aimed to characterise the HBV from Indigenous populations in Queensland and the Torres Strait Islands. METHODS: Serum samples were collected, with consent, from people within Queensland Indigenous communities prior to 1990 as part of the Queensland Health vaccination programme. Ethics approval was subsequently obtained to further characterise the HBV from 93 of these stored samples. HBV DNA was extracted and genotype was obtained from 82 samples. HBV full genome sequencing was carried out for a subset of 14 samples. RESULTS: Seventy-eight samples were identified as genotype C (2 × C12, 3 × C13 and 73 × C14), one sample as genotype A (A2) and three samples as genotype D (1 × D2, 1 × D3 and 1 × D4). The HBV/C sequences identified were most closely related to sequences isolated from Papua New Guinea and Indonesia (Papua Province). CONCLUSIONS: The HBV isolated from the Torres Strait Islanders was notably different to the HBV/C4 strain isolated from Indigenous people of mainland northern Australia, with no evidence of recombination. This reflects the differences in culture and origin between Torres Strait Islanders and mainland Indigenous people.

A phase 2, open-label, randomized, multiple-dose study evaluating Inarigivir in treatment-naïve patients with chronic hepatitis B.

BACKGROUND/AIMS: Novel agents acting against hepatitis B virus (HBV) are needed to improve HBsAg seroclearance or termed as 'functional cure'. Inarigivir (retinoic acid-inducible gene I agonist) has immunomodulatory and direct antiviral actions against HBV. We aimed to determine the safety and efficacy of Inarigivir for the treatment of HBV infection. PATIENTS/METHODS: 80 treatment-naïve patients were randomized in 4 ascending dose cohorts to receive 12 weeks of Inarigivir 25, 50, 100, 200 mg or placebo in a ratio of 4:1. All patients were then given tenofovir for another 12 weeks. RESULTS: Least squares (LS) mean reductions in HBV DNA from baseline increased with higher doses of Inarigivir (0.6116 in 25 mg and 1.5774 in 200 mg groups vs. 0.0352 in placebo group) (95% CI 0.9518-0.2011 and 1.921-1.1634 respectively). LS mean changes in HBV RNA and HBsAg from baseline ranged from -0.3856 to -0.5794 versus -0.1474 and -0.0956 to -0.1818 versus +0.0026 in Inarigivir-treated versus placebo groups respectively. During the tenofovir-treated period, LS mean reductions in HBsAg in the Inarigivir-treated groups ranged from 0.1709 to 0.3529 versus 0.1984 in the placebo group. Inarigivir-treated groups showed mean reductions in ALT from baseline between 23.3 and 33.8 versus 0.7 U/L in the placebo group. Treatment-emergent adverse events related to Inarigivir and placebo occurred in 4.7% and 6.3% patients respectively. CONCLUSIONS: Twelve-week Inarigivir up to 200 mg dose was associated with a reduction of HBV DNA, HBV RNA and antigen levels. A trend for greater HBsAg reduction was observed in Inarigivir pre-treated patients after switching to tenofovir.

Hepatitis B Virus Flares After Nucleot(s)ide Analogue Cessation Are Associated With Activation of Toll-Like Receptor Signaling Pathways.

BACKGROUND: We evaluated the patterns of peripheral Toll-like receptor (TLR) signaling activity and the expression of TLRs and natural killer (NK) cell activation in a cohort of patients experiencing severe hepatitis flares after stopping nucleot(s)ide analogues (NAs) therapy. METHODS: Samples were collected longitudinally from patients with chronic hepatitis B who were enrolled in a prospective study of NA discontinuation. Patients experiencing hepatitis flares were compared with patients with normal alanine aminotransferase. Peripheral blood mononuclear cells (PBMCs) were stimulated with TLR ligands and cytokine secretion in the cell culture supernatant measured. Expression of TLR2/4, NKG2D, NKp46, and triggering receptor expressed on myeloid cells 1 (TREM-1) on monocytes, NK, and NK-T cells was measured. RESULTS: Seventeen patients with severe reactivation hepatitis flares were compared to 12 nonflare patients. Hepatitis flares were associated with increased activity of TLR2-8 and TLR9 signaling in PBMCs at the time of peak flare compared to baseline. Hepatitis flares were also associated with (1) upregulation of TLR2 and (2) TREM-1 receptor expression on NK. There were no differences at baseline between flare patients and nonflare patients. CONCLUSIONS: Hepatitis flares off NA therapy have a significant innate inflammatory response with upregulation of TLR signaling on peripheral monocytes and TLR2 and TREM-1 expression on NK cells. This implicates the innate immune system in the immunopathogenesis of hepatitis B flares.

Stopping nucleot(s)ide analogues in non-cirrhotic HBeAg-negative chronic hepatitis B patients: HBsAg loss at 96 weeks is associated with low baseline HBsAg levels.

BACKGROUND AND AIMS: Current guidelines recommend long-term nucleot(s)ide analogue (NA) therapy for patients with HBeAg-negative chronic hepatitis B (CHB). However, disease remission has been described after stopping NA therapy, as well as HBsAg loss. METHODS: We performed a prospective multi-centre cohort study of stopping NA therapy. Inclusion criteria were HBeAg-negative CHB, the absence of cirrhosis and HBVDNA5× ULN occurred in 35 (32%); ALT flares were not associated with HBsAg loss. There were no unexpected safety issues. CONCLUSION: Virological reactivation was very common after stopping NA therapy and occurred earlier after stopping TDF versus ETV. The majority of patients had ALT <2× ULN at week 96, but only one-third achieved disease remission and HBsAg loss was rare. Very low HBsAg levels at baseline were uncommon but predicted for HBsAg loss and disease remission.

Origins and Evolution of the Primate Hepatitis B Virus.

Recent interest in the origins and subsequent evolution of the hepatitis B virus (HBV) has strengthened with the discovery of ancient HBV sequences in fossilized remains of humans dating back to the Neolithic period around 7,000 years ago. Metagenomic analysis identified a number of African non-human primate HBV sequences in the oldest samples collected, indicating that human HBV may have at some stage, evolved in Africa following zoonotic transmissions from higher primates. Ancestral genotype A and D isolates were also discovered from the Bronze Age, not in Africa but rather Eurasia, implying a more complex evolutionary and migratory history for HBV than previously recognized. Most full-length ancient HBV sequences exhibited features of inter genotypic recombination, confirming the importance of recombination and the mutation rate of the error-prone viral replicase as drivers for successful HBV evolution. A model for the origin and evolution of HBV is proposed, which includes multiple cross-species transmissions and favors subsequent recombination events that result in a pathogen and can successfully transmit and cause persistent infection in the primate host.

Analysis of Hepatitis B Virus Haplotype Diversity Detects Striking Sequence Conservation Across Genotypes and Chronic Disease Phase.

BACKGROUND AND AIMS: We conducted haplotype analysis of complete hepatitis B virus (HBV) genomes following deep sequencing from 368 patients across multiple phases of chronic hepatitis B (CHB) infection from four major genotypes (A-D), analyzing 4,110 haplotypes to identify viral variants associated with treatment outcome and disease progression. APPROACH AND RESULTS: Between 18.2% and 41.8% of nucleotides and between 5.9% and 34.3% of amino acids were 100% conserved in all genotypes and phases examined, depending on the region analyzed. Hepatitis B e antigen (HBeAg) loss by week 192 was associated with different haplotype populations at baseline. Haplotype populations differed across the HBV genome and CHB history, this being most pronounced in the precore/core gene. Mean number of haplotypes (frequency) per patient was higher in immune-active, HBeAg-positive chronic hepatitis phase 2 (11.8) and HBeAg-negative chronic hepatitis phase 4 (16.2) compared to subjects in the "immune-tolerant," HBeAg-positive chronic infection phase 1 (4.3, P< 0.0001). Haplotype frequency was lowest in genotype B (6.2, P< 0.0001) compared to the other genotypes (A = 11.8, C = 11.8, D = 13.6). Haplotype genetic diversity increased over the course of CHB history, being lowest in phase 1, increasing in phase 2, and highest in phase 4 in all genotypes except genotype C. HBeAg loss by week 192 of tenofovir therapy was associated with different haplotype populations at baseline. CONCLUSIONS: Despite a degree of HBV haplotype diversity and heterogeneity across the phases of CHB natural history, highly conserved sequences in key genes and regulatory regions were identified in multiple HBV genotypes that should be further investigated as targets for antiviral therapies and predictors of treatment response.

The evolution and clinical impact of hepatitis B virus genome diversity.

The global burden of hepatitis B virus (HBV) is enormous, with 257 million persons chronically infected, resulting in more than 880,000 deaths per year worldwide. HBV exists as nine different genotypes, which differ in disease progression, natural history and response to therapy. HBV is an ancient virus, with the latest reports greatly expanding the host range of the Hepadnaviridae (to include fish and reptiles) and casting new light on the origins and evolution of this viral family. Although there is an effective preventive vaccine, there is no cure for chronic hepatitis B, largely owing to the persistence of a viral minichromosome that is not targeted by current therapies. HBV persistence is also facilitated through aberrant host immune responses, possibly due to the diverse intra-host viral populations that can respond to host-mounted and therapeutic selection pressures. This Review summarizes current knowledge on the influence of HBV diversity on disease progression and treatment response and the potential effect on new HBV therapies in the pipeline. The mechanisms by which HBV diversity can occur both within the individual host and at a population level are also discussed.

Glycoengineered hepatitis B virus-like particles with enhanced immunogenicity.

Virus-like particles (VLP) represent biological platforms for the development of novel products such as vaccines and delivery platforms for foreign antigenic sequences. VLPs composed of the small surface antigen (HBsAgS) derived from the hepatitis B virus (HBV) are the immunogenic components of a licensed, preventative vaccine, which contains aluminum hydroxide as adjuvant. Herein, we report that glycoengineering of N-glycosylated HBsAgS to generate hyper-glycosylated VLPs display an enhanced immunogenicity relative to the wild type (WT) HBsAgS VLPs when expressed in FreeStyle HEK 293F cells. Comparative mass spectrometry-based N-glycan profiling, gel electrophoresis, and immunoassays demonstrated that WT and hyper-glycosylated HBsAgS VLPs contain the same type and distribution of N-glycan structures, but the latter shows a higher glycan abundance per protein mass. The antigenic integrity of the modified VLPs was also shown to be retained. To assess whether hyper-glycosylated VLPs induce an enhanced immune response in the presence of the adjuvant aluminum hydroxide, the anti-HBV surface antigen (anti-HBsAgS) antibody response was monitored in BALB/c mice, subcutaneously injected with different VLP derivatives. In the absence and presence of adjuvant, hyper-glycosylated VLPs showed an enhanced immunogenicity compared to WT VLPs. The ability of hyper-glycosylated VLPs to promote potent anti-HBsAgS immune responses compared to VLPs with a native N-glycan level as well as non-glycosylated, yeast-derived HBsAgS VLPs opens exciting avenues for generating more efficacious VLP-based vaccines against hepatitis B and improved HBsAgS VLP carrier platforms using glycoengineering.

RNA Interference Therapy With ARC-520 Results in Prolonged Hepatitis B Surface Antigen Response in Patients With Chronic Hepatitis B Infection.

BACKGROUND AND AIMS: ARC-520, the first an RNA interference (RNAi) therapeutic, was designed to reduce all RNA transcripts derived from covalently closed circular DNA, leading to a reduction in viral antigens and hepatitis B virus (HBV) DNA. APPROACH AND RESULTS: We aimed to evaluate the depth of hepatitis B surface antigen (HBsAg) decline in response to multiple doses of ARC-520 compared to placebo (PBO) in two randomized, multicenter studies in nucleoside/nucleotide analogue reverse-transcriptase inhibitor (NUC)-experienced patients with hepatitis B early antigen (HBeAg)-negative (E-neg) or HBeAg-positive (E-pos) disease. A total of 58 E-neg and 32 E-pos patients were enrolled and received four monthly doses of PBO (n = 20 E-neg, 11 E-pos), 1 mg/kg ARC-520 (n = 17 E-neg, 10 E-pos), or 2 mg/kg ARC-520 (n = 21 E-neg, 11 E-pos) concomitantly with NUC. HBsAg change from baseline to 30 days after the last ARC-520 dose were compared to PBO. Both E-neg and E-pos high-dose groups significantly reduced HBsAg compared to PBO, with mean reductions of 0.38 and 0.54 log IU/mL, respectively. HBsAg reductions persisted for approximately 85 days and >85 days after the last dose in E-neg and E-pos patients, respectively. The low-dose groups did not reach statistical significance in either study. E-pos patients showed a dose-dependent reduction in HBeAg from baseline. Mean maximum reduction was 0.23 and 0.69 log Paul Ehrlich IUs/mL in the low-dose and high dose ARC-520 groups respectively. ARC-520 was well tolerated, with only two serious adverse events of pyrexia possibly related to study drug observed. CONCLUSIONS: ARC-520 was active in both E-neg and E-pos, NUC-experienced HBV patients; but absolute HBsAg reductions were moderate, possibly due to expression of HBsAg from integrated HBV DNA, indicating the need for RNAi therapeutics that can target viral transcripts regardless of origin.

Hepatitis B virus infection.

Hepatitis B virus (HBV) is a hepatotropic virus that can establish a persistent and chronic infection in humans through immune anergy. Currently, 3.5% of the global population is chronically infected with HBV, although the incidence of HBV infections is decreasing owing to vaccination and, to a lesser extent, the use of antiviral therapy to reduce the viral load of chronically infected individuals. The course of chronic HBV infection typically comprises different clinical phases, each of which potentially lasts for decades. Well-defined and verified serum and liver biopsy diagnostic markers enable the assessment of disease severity, viral replication status, patient risk stratification and treatment decisions. Current therapy includes antiviral agents that directly act on viral replication and immunomodulators, such as interferon therapy. Antiviral agents for HBV include reverse transcriptase inhibitors, which are nucleoside or nucleotide analogues that can profoundly suppress HBV replication but require long-term maintenance therapy. Novel compounds are being actively investigated to achieve the goal of HBV surface antigen seroclearance (functional cure), a serological state that is associated with a higher remission rate (thus, no viral rebound) after treatment cessation and a lower rate of cirrhosis and hepatocellular carcinoma. This Primer addresses several aspects of HBV infection, including epidemiology, immune pathophysiology, diagnosis, prevention and management.

Mother-to-child transmission of hepatitis B: Examining viral cut-offs, maternal HBsAg serology and infant testing.

BACKGROUND & AIMS: Antipartum antiviral therapy in the setting of high viral load is recommended to prevent mother-to-child transmission of hepatitis B although recommended viral load cut-offs vary. Quantitative HBsAg has been proposed as an alternative screening strategy to identify high viral load in this setting. Guidelines suggest testing all infants for vaccine response and infection. We set out to re-examine viral load cut-offs; the predictive value of quantitative HBsAg and the need for follow-up infant testing in our cohort. METHODS: A retrospective cohort study of 469 HBsAg positive mother-baby pairs from 2 tertiary hospitals in Sydney was performed. Antiviral therapy (lamivudine or tenofovir disoproxil fumarate) was offered to women with viral load ≥6 log10  IU/mL (high) from 32 weeks gestation. Transmission and vaccine response was analysed according to viral load. The utility of quantitative HBsAg in identifying high viral load was examined. RESULTS: Mother-to-child transmission only occurred in setting of high viral load, in 0.85% (1/117) of those who received antiviral therapy and in 8.66% (2/23) of those who chose not to. Quantitative HBsAg did not accurately identify high-risk mothers HBV DNA ≥6 log10  IU/mL. Successful infant vaccine response was 98.7% overall, and 99.4% when viral load was <6 log10  IU/mL. CONCLUSION: Antiviral therapy initiated at 32 weeks when maternal viral load is ≥6 log10  IU/mL almost completely abrogates transmission. Quantitative HBsAg does not reliably predict high viral load. When maternal viral load is <6 log10  IU/mL, high vaccine efficacy and zero transmission suggests testing infants is of little value.

RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.

Chronic hepatitis B virus (HBV) infection is a major health concern worldwide, frequently leading to liver cirrhosis, liver failure, and hepatocellular carcinoma. Evidence suggests that high viral antigen load may play a role in chronicity. Production of viral proteins is thought to depend on transcription of viral covalently closed circular DNA (cccDNA). In a human clinical trial with an RNA interference (RNAi)-based therapeutic targeting HBV transcripts, ARC-520, HBV S antigen (HBsAg) was strongly reduced in treatment-naïve patients positive for HBV e antigen (HBeAg) but was reduced significantly less in patients who were HBeAg-negative or had received long-term therapy with nucleos(t)ide viral replication inhibitors (NUCs). HBeAg positivity is associated with greater disease risk that may be moderately reduced upon HBeAg loss. The molecular basis for this unexpected differential response was investigated in chimpanzees chronically infected with HBV. Several lines of evidence demonstrated that HBsAg was expressed not only from the episomal cccDNA minichromosome but also from transcripts arising from HBV DNA integrated into the host genome, which was the dominant source in HBeAg-negative chimpanzees. Many of the integrants detected in chimpanzees lacked target sites for the small interfering RNAs in ARC-520, explaining the reduced response in HBeAg-negative chimpanzees and, by extension, in HBeAg-negative patients. Our results uncover a heretofore underrecognized source of HBsAg that may represent a strategy adopted by HBV to maintain chronicity in the presence of host immunosurveillance. These results could alter trial design and endpoint expectations of new therapies for chronic HBV.

The virological aspects of hepatitis B.

Human hepatitis B virus (HBV) is a hepatotropic virus that is responsible for a significant burden of disease, causing liver disease and hepatocellular carcinoma. It is a small DNA virus with a replication strategy that is similar to that of a retrovirus. HBV is prone to mutagenesis and under the influence of diverse selection pressures, has evolved into a pool of quasispecies, genotypes and mutants, which confers a significant survival advantage. The genome is small, circular, and compact but has a complex replication strategy. The viral life cycle involves the formation of a covalently closed circular DNA (cccDNA), which is organized into a minichromosome that is the template for the synthesis of viral mRNA. HBV DNA (double-stranded linear form) can also integrate into the host genome, ensuring lifelong persistence of the virus. To date, despite great advances in therapeutics, once HBV is chronically established, it is incurable. This is by virtue of many aspects of its virological structure and viral life cycle. In this review, we aim to discuss important aspects of the virology of HBV with a focus on clinical implications.

Safety, Tolerability, and Pharmacokinetics of ARC-520 Injection, an RNA Interference-Based Therapeutic for the Treatment of Chronic Hepatitis B Virus Infection, in Healthy Volunteers.

ARC-520 Injection, an RNA interference drug for the treatment of hepatitis B that targets cccDNA-derived viral mRNA transcripts with high specificity, effectively reduces the production of viral proteins and HBV DNA. In this phase 1 randomized, double-blind, placebo-controlled study, 54 healthy volunteers (half male, half female) received a single, intravenous dose of 0.01-4.0 mg/kg ARC-520 Injection (n = 36) or placebo (n = 18). Assessments included safety, tolerability, pharmacokinetics, and pharmacodynamics (cytokines and complement). Pharmacokinetics of the siRNA and peptide excipient components contained in ARC-520 Injection showed a relatively short half-life of 3-5 and 8-10 hours, respectively. Dose exposure linearity was demonstrated within the dose range. ARC-520 Injection was well tolerated, with adverse-event frequency the same as placebo and no serious adverse events. ARC-520 Injection was initially found to induce histamine release through mast cell degranulation, resulting in 2 moderate hypersensitivity reactions. However, after initiation of pretreatment with oral antihistamine, no further hypersensitivity reactions occurred. Low-level, transient complement induction and sporadic, mild, and transient elevations of several cytokines were observed but not associated with any symptoms. ARC-520 Injection showed a favorable tolerability profile in this single-dose study in healthy volunteers. Oral antihistamine pretreatment is recommended in the future to offset mast cell degranulation stimulation.

In Vitro Studies Show that Sequence Variability Contributes to Marked Variation in Hepatitis B Virus Replication, Protein Expression, and Function Observed across Genotypes.

The hepatitis B virus (HBV) exists as 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes. Marked differences in HBV natural history, disease progression and treatment response are exhibited by many of these genotypes and subtypes. For example, HBV genotype C is associated with later hepatitis B e antigen (HBeAg) seroconversion and high rates of liver cancer compared to other HBV genotypes, whereas genotype A2 is rarely associated with HBeAg-negative disease or liver cancer. The reasons for these and other differences in HBV natural history are yet to be determined but could in part be due to sequence differences in the HBV genome that alter replicative capacity and/or gene expression. Direct comparative studies on HBV replication and protein expression have been limited to date due largely to the absence of infectious HBV cDNA clones for each of the HBV genotypes present in the same genetic arrangement. We have produced replication-competent infectious cDNA clones of the most common subtypes of genotypes A to D, namely, A2, B2, C2, D3, and the minor strain J, and compared their HBV replication phenotype using transient-transfection models. We identified striking differences in HBV replicative capacity as well as HBeAg and surface (HBsAg) protein expression across genotypes, which may in part be due to sequence variability in regulatory regions of the HBV genome. Functional analysis showed that sequence differences in the major upstream regulatory region across genotypes impacted promoter activity. IMPORTANCE: There have been very few studies directly comparing the replication phenotype of different HBV genotypes, for which there are marked differences in natural history and disease progression worldwide. We have generated replication-competent 1.3-mer cDNA clones of the major genotypes A2, B2, C2, and D3, as well as a recently identified strain J, and identified striking differences in replicative capacity and protein expression that may contribute to some of the observed differences in HBV natural history observed globally.

New perspectives on the hepatitis B virus life cycle in the human liver.

The central role of the transcriptional template of the hepatitis B virus (HBV), covalently closed circular DNA (cccDNA), has been difficult to study in patients with chronic hepatitis B (CHB) infection. In this issue of the JCI, Zhang and colleagues reveal a mosaic distribution of viral antigens and nucleic acids and a mismatch between HBV cccDNA, RNA, and expression of the hepatitis B surface antigen (HBsAg). These unusual patterns varied over the natural history of CHB, prompting the authors to propose a new three-stage model of the HBV life cycle at the single-cell level.

Synthetic RNAi triggers and their use in chronic hepatitis B therapies with curative intent.

Current therapies for chronic hepatitis B virus infection (CHB) - nucleos(t)ide analogue reverse transcriptase inhibitors and interferons - result in low rates of functional cure defined as sustained off-therapy seroclearance of hepatitis B surface antigen (HBsAg). One likely reason is the inability of these therapies to consistently and substantially reduce the levels of viral antigen production. Accumulated evidence suggests that high serum levels of HBsAg result in exhaustion of the host immune system, rendering it unable to mount the effective antiviral response required for HBsAg clearance. New mechanistic approaches are required to produce high rates of HBsAg seroclearance in order to greatly reduce off-treatment disease progression. Already shown to be a clinically viable means of reducing gene expression in a number of other diseases, therapies based on RNA interference (RNAi) can directly target hepatitis B virus transcripts with high specificity, profoundly reducing the production of viral proteins. The fact that the viral RNA transcripts contain overlapping sequences means that a single RNAi trigger can result in the degradation of all viral transcripts, including all messenger RNAs and pregenomic RNA. Advances in the design of RNAi triggers have increased resistance to degradation and reduced nonspecific innate immune stimulation. Additionally, new methods to effectively deliver the trigger to liver hepatocytes, and specifically to the cytoplasmic compartment, have resulted in increased efficacy and tolerability. An RNAi-based drug currently in clinical trials is ARC-520, a dynamic polyconjugate in which the RNAi trigger is conjugated to cholesterol, which is coinjected with a hepatocyte-targeted, membrane-active peptide. Phase 2a clinical trial results indicate that ARC-520 was well tolerated and resulted in significant, dose-dependent reduction in HBsAg for up to 57days in CHB patients. RNAi-based therapies may play an important role in future therapeutic regimes aimed at improving HBsAg seroclearance and eliminating the need for lifelong therapy. This paper 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 B."

Chronic hepatitis B: Virology, natural history, current management and a glimpse at future opportunities.

The host immune system plays an important role in chronic hepatitis B (CHB), both in viral clearance and hepatocellular damage. Advances in our understanding of the natural history of the disease have led to redefining the major phases of infection, with the "high replicative, low inflammatory" phase now replacing what was formerly termed the "immune tolerant" phase, and the "nonreplicative phase" replacing what was formerly termed the "inactive carrier" phase. As opposed to the earlier view that HBV establishes chronic infection by exploiting the immaturity of the neonate's immune system, new findings on trained immunity show that the host is already somewhat "matured" following birth, and is actually very capable of responding immunologically, potentially altering future hepatitis B treatment strategies. While existing therapies are effective in reducing viral load and necroinflammation, often restoring the patient to near-normal health, they do not lead to a cure except in very rare cases and, in many patients, viremia rebounds after cessation of treatment. Researchers are now challenged to devise therapies that will eliminate infection, with a particular focus on eliminating the persistence of viral cccDNA in the nuclei of hepatocytes. In the context of chronic hepatitis B, new definitions of 'cure' are emerging, such as 'functional' and 'virological' cure, defined by stable off-therapy suppression of viremia and antigenemia, and the normalization of serum ALT and other liver-related laboratory tests. Continued advances in the understanding of the complex biology of chronic hepatitis B have resulted in the development of new, experimental therapies targeting viral and host factors and pathways previously not accessible to therapy, approaches which may lead to virological cures in the near term and functional cures upon long term follow-up. 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 B."

Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

BACKGROUND: The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. METHODS: Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. RESULTS: In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-ß and α-SMA expression in CCl4 treated mice compared to controls. CONCLUSION: We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.