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

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

Constrained evolution of overlapping genes in viral host adaptation: Acquisition of glycosylation motifs in hepadnaviral precore/core genes.

Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes.

Host cell-dependent late entry step as determinant of hepatitis B virus infection.

Hepatitis B virus (HBV) has a highly restricted host range and cell tropism. Other than the human sodium taurocholate cotransporting polypeptide (huNTCP), the HBV entry receptor, host determinants of HBV susceptibility are poorly understood. Woodchucks are naturally infected with woodchuck hepatitis virus (WHV), closely related to HBV, but not with HBV. Here, we investigated the capabilities of woodchuck hepatic and human non-hepatic cell lines to support HBV infection. DNA transfection assays indicated that all cells tested supported both HBV and WHV replication steps post entry, including the viral covalently closed circular DNA (cccDNA) formation, which is essential for establishing and sustaining infection. Ectopic expression of huNTCP rendered one, but not the other, woodchuck hepatic cell line and the non-hepatic human cell line competent to support productive HBV entry, defined here by cccDNA formation during de novo infection. All huNTCP-expressing cell lines tested became susceptible to infection with hepatitis D virus (HDV) that shares the same entry receptor and initial steps of entry with HBV, suggesting that a late entry/trafficking step(s) of HBV infection was defective in one of the two woodchuck cell lines. In addition, the non-susceptible woodchuck hepatic cell line became susceptible to HBV after fusion with human hepatic cells, suggesting the lack of a host cell-dependent factor(s) in these cells. Comparative transcriptomic analysis of the two woodchuck cell lines revealed widespread differences in gene expression in multiple biological processes that may contribute to HBV infection. In conclusion, other than huNTCP, neither human- nor hepatocyte-specific factors are essential for productive HBV entry. Furthermore, a late trafficking step(s) during HBV infection, following the shared entry steps with HDV and before cccDNA formation, is subject to host cell regulation and thus, a host determinant of HBV infection.

Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection.

BACKGROUND AND AIMS: The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints. APPROACH AND RESULTS: Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA. CONCLUSIONS: In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.

Toll-Like Receptor 8 Agonist GS-9688 Induces Sustained Efficacy in the Woodchuck Model of Chronic Hepatitis B.

BACKGROUND AND AIMS: GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus. APPROACH AND RESULTS: WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS-9688. CONCLUSIONS: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.

4-Oxooctahydroquinoline-1(2H)-carboxamides as hepatitis B virus (HBV) capsid core protein assembly modulators.

Hepatitis B virus (HBV) core protein, the building block of the HBV capsid, plays multiple roles in viral replication, and is an attractive target for development of antiviral agents with a new mechanism of action. In addition to the heteroaryldihydropyrimidines (HAPs), sulfamoylbenzamides (SBAs), dibenzothiazepine derivatives (DBTs), and sulfamoylpyrrolamides (SPAs) that inhibit HBV replication by modulation of viral capsid assembly and are currently under clinical trials for the treatment of chronic hepatitis B (CHB), other chemical structures with activity to modulate HBV capsid assembly have also been explored. Here we describe our continued optimization of a benzamide originating from our high throughput screening. A new bicyclic carboxamide lead featuring an electron deficient non-planar core structure was discovered. Evaluations of its ADMET (absorption, distribution, metabolism, excretion and toxicity) and pharmacokinetic (PK) profiles demonstrate improved metabolic stability and good bioavailability.

Innate and adaptive immunity associated with resolution of acute woodchuck hepatitis virus infection in adult woodchucks.

Viral and/or host factors that are directly responsible for the acute versus chronic outcome of hepatitis B virus (HBV) infection have not been identified yet. Information on immune response during the early stages of HBV infection in humans is mainly derived from blood samples of patients with acute hepatitis B (AHB), which are usually obtained after the onset of clinical symptoms. Features of intrahepatic immune response in these patients are less studied due to the difficulty of obtaining multiple liver biopsies. Woodchuck hepatitis virus (WHV) infection in woodchucks is a model for HBV infection in humans. In the present study, five adult woodchucks were experimentally infected with WHV and then followed for 18 weeks. Blood and liver tissues were frequently collected for assaying markers of WHV replication and innate and adaptive immune responses. Liver tissues were further analyzed for pathological changes and stained for important immune cell subsets and cytokines. The increase and subsequent decline of viral replication markers in serum and liver, the elicitation of antibodies against viral proteins, and the induction of virus-specific T-cell responses indicated eventual resolution of acute WHV infection in all animals. Intrahepatic innate immune makers stayed unchanged immediately after the infection, but increased markedly during resolution, as determined by changes in transcript levels. The presence of interferon-gamma and expression of natural killer (NK) cell markers suggested that a non-cytolytic response mechanism is involved in the initial viral control in liver. This was followed by the expression of T-cell markers and cytolytic effector molecules, indicating the induction of a cytolytic response mechanism. Parallel increases in regulatory T-cell markers suggested that this cell subset participates in the overall immune cell infiltration in liver and/or has a role in regulating AHB induced by the cytolytic response mechanism. Since the transcript levels of immune cell markers in blood, when detectable, were lower than in liver, and the kinetics, except for NK-cells and interferon-gamma, did not correlate well with their intrahepatic expression, this further indicated enrichment of immune cells within liver. Conclusion: The coordinated interplay of innate and adaptive immunity mediates viral clearance in the woodchuck animal model of HBV infection. The initial presence of NK-cell associated interferon-gamma response points to an important role of this cytokine in HBV resolution.

Synthesis of 4-oxotetrahydropyrimidine-1(2H)-carboxamides derivatives as capsid assembly modulators of hepatitis B virus.

We report herein the synthesis and evaluation of phenyl ureas derived from 4-oxotetrahydropyrimidine as novel capsid assembly modulators of hepatitis B virus (HBV). Among the derivatives, compound 27 (58031) and several analogs showed an activity of submicromolar EC50 against HBV and low cytotoxicities (>50 µM). Structure-activity relationship studies revealed a tolerance for an additional group at position 5 of 4-oxotetrahydropyrimidine. The mechanism study indicates that compound 27 (58031) is a type II core protein allosteric modulator (CpAMs), which induces core protein dimers to assemble empty capsids with fast electrophoresis mobility in native agarose gel. These compounds may thus serve as leads for future developments of novel antivirals against HBV.

Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

Prevention of liver tumor formation in woodchucks with established hepatocellular carcinoma by treatment with cationic liposome-DNA complexes.

BACKGROUND: Approximately 250 million people worldwide are chronically infected with hepatitis B virus (HBV) and more than half of the hepatocellular carcinoma (HCC) cases are attributed to this infection. As HCC has a high mortality rate, and current treatment options are remarkably limited, the development of new therapeutic treatment strategies is warranted. METHODS: In this study, woodchucks infected with woodchuck hepatitis virus (WHV), and with pre-existing liver tumors, were used as a model to investigate if complexes of cationic liposomes and non-coding DNA (JVRS-100) were effective in treatment of HCC. RESULTS: It was observed that the high serum viral load that is present in a typical chronic WHV infection (i.e., approximately 100-fold higher than human viral loads) results in immune suppression and resistance to treatment with JVRS-100. Treatment of woodchucks with lower serum viral load that more closely matched with the viral load usually seen in human HBV infection appears a better model for immunotherapeutic development based on the responsiveness to JVRS-100 treatment. In the latter case, marked declines in WHV DNA and WHV surface antigen were determined over the 12-week treatment period and WHV markers stayed suppressed during most time points of the 12-week follow-up period. Even more remarkably, the formation of new liver tumors was not observed in woodchucks treated with a well-tolerated dose of JVRS-100, as compared to several new tumors that developed in vehicle-treated control animals. CONCLUSIONS: Although there was little decrease in the volumes of the liver tumors existing at the time of treatment, it is generally accepted that preventing the spread and metastasis of almost always fatal cancers such as HCC and thus, reducing it to a chronic and treatable disease can also be a successful therapeutic approach. The results in woodchucks warrant the investigation of JVRS-100 as an intervention to prevent liver cancer in patients chronically infected with HBV and at high risk for HCC development.

Superinfection with woodchuck hepatitis virus strain WHVNY of livers chronically infected with strain WHV7.

UNLABELLED: The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all), virus superinfection is an unlikely event, and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Superinfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superinfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superinfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superinfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superinfection. The results are consistent with the occurrence of continuous but inefficient hepadnavirus cell-to-cell spread and superinfection during chronic infection and suggest that the replication space occupied by the superinfecting hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic hepadnavirus infection. IMPORTANCE: This study aimed to better understand the determinants of the maintenance of chronic hepadnavirus infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) hepadnavirus superinfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic hepadnavirus infection. The significance of the continuous but limited hepadnavirus spread and superinfection for the maintenance of the chronic state of infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic hepadnavirus infection.

Infection Patterns Induced in Naive Adult Woodchucks by Virions of Woodchuck Hepatitis Virus Collected during either the Acute or Chronic Phase of Infection.

UNLABELLED: The infectivity of hepadnavirus virions produced during either acute or chronic stages of infection was compared by testing the ability of the virions of woodchuck hepatitis virus (WHV) to induce productive acute infection in naive adult woodchucks. Serum WHV collected during acute infection was compared to virions harvested from WHV-infected woodchucks during either (i) early chronic infection, when WHV-induced hepatocellular carcinoma (HCC) was not yet developed, or (ii) late chronic infection, when established HCC was terminal. All tested types of WHV inoculum were related, because they were collected from woodchucks that originally were infected with standardized WHV7 inoculum. Despite the individual differences between animals, the kinetics of accumulation of serum relaxed circular DNA of WHV demonstrated that the virions produced during early or late chronic infection are fully capable of inducing productive acute infection with long-lasting high viremia. These findings were further supported by the analysis of such intrahepatic markers of WHV infection as replicative intermediate DNA, covalently closed circular DNA, pregenomic RNA, and the percentage of WHV core antigen-positive hepatocytes measured at several time points over the course of 17.5 weeks after the inoculation. In addition, the observed relationship between the production of antibodies against WHV surface antigens and parameters of WHV infection appears to be complex. Taken together, the generated data suggest that in vivo hepadnavirus virions produced during different phases of chronic infection did not demonstrate any considerable deficiencies in infectivity compared to that of virions generated during the acute phase of infection. IMPORTANCE: The generated data suggest that infectivity of virions produced during the early or late stages of chronic hepadnavirus infection is not compromised. Our novel results provided several lines of further evidence supporting the idea that during the state of chronic infection in vivo, the limitations of hepadnavirus cell-to-cell spread/superinfection (observed recently in the woodchuck model) are not due to the diminished infectivity of the virions circulating in the blood and likely are (i) related to the properties of hepatocytes (i.e., their capacity to support hepadnavirus infection/replication) and (ii) influenced by the immune system. The obtained results further extend the understanding of the mechanisms regulating the persistence of hepadnavirus infection. Follow-up studies that will further investigate hepadnavirus cell-to-cell spread as a potential regulator of the chronic state of the infection are warranted.

Liver-directed gene therapy of chronic hepadnavirus infection using interferon alpha tethered to apolipoprotein A-I.

BACKGROUND & AIMS: Current hepatitis B virus (HBV) management is challenging as treatment with nucleos(t)ide analogues needs to be maintained indefinitely and because interferon (IFN)-α therapy is associated with considerable toxicity. Previously, we showed that linking IFNα to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFNα. METHODS: Here, we analyse the antiviral potential of an adeno-associated vector encoding IFNα fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFNα (AAV-IFN) in two animal models of chronic hepadnavirus infection. RESULTS: In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic hepatitis virus (WHV) infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA), experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN, while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchucks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. CONCLUSIONS: Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult to treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogues represents a promising approach for the treatment of HBV infection in highly viremic patients.

Sustained efficacy and seroconversion with the Toll-like receptor 7 agonist GS-9620 in the Woodchuck model of chronic hepatitis B.

BACKGROUND & AIMS: New therapies for chronic hepatitis B (CHB) are urgently needed since current treatments rarely lead to cure. We evaluated whether the oral small molecule toll-like receptor (TLR7) agonist GS-9620 could induce durable antiviral efficacy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to human hepatitis B virus (HBV). METHODS: After evaluating the pharmacokinetics, pharmacodynamics and tolerability of oral GS-9620 in uninfected woodchucks, adult woodchucks chronically infected with WHV (n = 7 per group) were dosed with GS-9620 or placebo for 4 or 8 weeks with different treatment schedules. RESULTS: GS-9620 treatment induced rapid, marked and sustained reduction in serum viral DNA (mean maximal 6.2log10 reduction), and hepatic WHV DNA replicative intermediates, WHV cccDNA and WHV RNA, as well as loss of detectable serum WHV surface antigen (WHsAg). GS-9620 treatment also induced a sustained antibody response against WHsAg in a subset of animals. Strikingly, treatment reduced the incidence of hepatocellular carcinoma (HCC) from 71% in the placebo group to 8% in GS-9620-treated woodchucks with sustained viral load reduction. GS-9620 treatment was associated with reversible increases in serum liver enzymes and thrombocytopenia, and induced intrahepatic CD8(+) T cell, NK cell, B cell and interferon response transcriptional signatures. CONCLUSIONS: The data demonstrate that short duration, finite treatment with the oral TLR7 agonist GS-9620 can induce a sustained antiviral response in the woodchuck model of CHB, and support investigation of this compound as a therapeutic approach to attain a functional cure in CHB patients.

Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and release of infectious hepatitis delta virus particles.

UNLABELLED: A natural subviral agent of human hepatitis B virus (HBV), hepatitis delta virus (HDV), requires only the envelope proteins from HBV in order to maintain persistent infection. HBV surface antigens (HBsAgs) can be produced either by HBV replication or from integrated HBV DNA regardless of replication. The functional properties of the integrant-generated HBsAgs were examined using two human hepatocellular carcinoma-derived cell lines, Hep3B and PLC/PRF/5, that contain HBV integrants but do not produce HBV virions and have no signs of HBV replication. Both cell lines were able to support HDV replication and assembly/egress of HDV virions. Neither of the cell lines was able to produce substantial amounts of the pre-S1-containing HDV particles. HDV virions assembled in PLC/PRF/5 cells were able to infect primary human hepatocytes, while Hep3B-derived HDV appeared to be noninfectious. These results correlate with the findings that the entire open reading frame (ORF) for the large (L) envelope protein that is essential for infectivity is present on HBV RNAs from PLC/PRF/5 cells, while an L protein ORF that was truncated and fused to inverted precore sequences was found using RNAs from Hep3B cells. This study demonstrates for the first time that at least some of the HBV DNA sequence naturally integrated during infection can produce functional small and large envelope proteins capable of the formation of infectious HDV virions. Our data indicate that in vivo chronic HDV infection can persist in the absence of HBV replication (or when HBV replication is profoundly suppressed) if functional envelope proteins are supplied from HBV integrants. IMPORTANCE: The study addresses the unique mechanism of HDV persistence in the absence of ongoing HBV replication, advances our understanding of HDV-HBV interactions, and supports the implementation of treatments directly targeting HDV for HDV/HBV-infected individuals.

Support of the infectivity of hepatitis delta virus particles by the envelope proteins of different genotypes of hepatitis B virus.

UNLABELLED: This study examined how the envelope proteins of 25 variants of hepatitis B virus (HBV) genotypes A to I support hepatitis delta virus (HDV) infectivity. The assembled virions bore the same HDV ribonucleoprotein and differed only by the HBV variant-specific envelope proteins coating the particles. The total HDV yields varied within a 122-fold range. A residue Y (position 374) in the HDV binding site was identified as critical for HDV assembly. Virions that bound antibodies, which recognize the region that includes the HBV matrix domain and predominantly but not exclusively immunoprecipitate the PreS1-containing virions, were termed PreS1*-HDVs. Using in vitro infection of primary human hepatocytes (PHH), we measured the specific infectivity (SI), which is the number of HDV genomes/cell produced by infection and normalized by the PreS1*-MOI, which is the multiplicity of infection that reflects the number of PreS1*-HDVs per cell in the inoculum used. The SI values varied within a 160-fold range and indicated a probable HBV genotype-specific trend of D > B > E > A in supporting HDV infectivity. Three variants, of genotypes B, C, and D, supported the highest SI values. We also determined the normalized index (NI) of infected PHH, which is the percentage of HDV-infected hepatocytes normalized by the PreS1*-MOI. Comparison of the SI and NI values revealed that, while a particular HBV variant may facilitate the infection of a relatively significant fraction of PHH, it may not always result in a considerable number of genomes that initiated replication after entry. The potential implications of these findings are discussed in the context of the mechanism of attachment/entry of HBV and HDV. IMPORTANCE: The study advances the understanding of the mechanisms of (i) attachment and entry of HDV and HBV and (ii) transmission of HDV infection/disease.

Identification of an intrahepatic transcriptional signature associated with self-limiting infection in the woodchuck model of hepatitis B.

UNLABELLED: The woodchuck model of hepatitis B virus (HBV) infection displays many characteristics of human infection and has particular value for characterizing the host immune responses during the development of chronic infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional profiles of neonatal woodchucks with self-limiting woodchuck hepatitis virus (WHV) infection to those woodchucks progressing to persistent WHV infection. This revealed that WHV does not induce significant intrahepatic gene expression changes during the early-acute stage of infection (8 weeks), suggesting it is a stealth virus. At the mid-acute phase of infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T-cell signature. Strikingly, this was accompanied by high-level expression of PD-1 and various other inhibitory T-cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. In contrast to the expression of perforin and other cytotoxic effector genes, the interferon-γ (IFN-γ) signaling response in the mid-acute phase was comparable to that in chronically infected adult animals. The absence of a strong IFN-α/ß transcriptional response indicated that type I IFN is not a critical mediator of self-limiting infection. Nevertheless, a number of antiviral genes, including viperin, were differentially expressed during resolving infection, suggesting that a subset of IFN-stimulated genes (ISG) may play a role in the control of WHV replication. CONCLUSION: We identified new immune pathways associated with the clearance of hepadnavirus infection revealing novel molecular targets with potential for the therapeutic treatment of chronic hepatitis B.

Expression of matrix metalloproteinases and their inhibitors in the woodchuck model of hepatocellular carcinoma.

Matrix metalloproteinases (MMPs) play a central role in tumor invasion and metastasis. Increased expression of MMPs occurs during development of hepatocellular carcinoma (HCC) in humans following infection with hepatitis B virus (HBV). Woodchucks are used as an animal model for hepadnavirus-induced HCC. All woodchucks infected chronically with woodchuck hepatitis virus (WHV), a virus that is closely related to HBV, develop HCC. In the present study MMPs and related molecules were investigated in woodchucks to better understand the mechanisms of extracellular matrix remodeling in HCC. Three groups of samples were studied: liver and HCC tissues from animals infected with WHV and age- and gender-matched normal liver from animals not infected with WHV. New partial gene sequences for woodchuck MMP-2, MMP-7, and MMP-9 as well as their inhibitors NGAL, TIMP-1, and TIMP-2 were identified and used for determination of expression levels in liver and HCC by qRT-PCR. Compared to liver of WHV-naïve woodchucks, high levels of MMP-1, MMP-2, MMP-7, NGAL, and TIMP-1 were detected in liver of animals infected with WHV. However, no differences were found for TIMP-2. MMP-9 expression was higher in HCC than in liver of animals not infected with WHV. Immunohistochemical staining demonstrated that MMP-9 immunoreactivity was most intense in HCC, correlating with the progression of liver disease. Upregulation of MMP-9 in HCC was confirmed by Western blotting and zymography analysis. Furthermore, the activity of woodchuck MMPs was suppressed by BiPS, a common inhibitor of mammalian MMPs. These results suggest the use of MMP inhibitors as a potential HCC treatment strategy that could be explored in woodchucks.

Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks.

UNLABELLED: Hepatitis delta virus (HDV) is a natural subviral agent of human hepatitis B virus (HBV). HDV enhances liver damage during concomitant infection with HBV. The molecular pathogenesis of HDV infection remains poorly understood. To advance our understanding of the relationship between HDV infection and liver cancer, it was determined whether HDV could infect in vivo the cells of hepadnavirus-induced hepatocellular carcinoma (HCC). Woodchucks (Marmota monax) that were chronically infected with HBV-related woodchuck hepatitis virus (WHV) and already developed HCCs were used as an experimental model. The locations of HCCs within the livers were determined using ultrasound imaging followed by open surgery. One week after surgery the WHV carrier woodchucks were superinfected with WHV-enveloped HDV (wHDV). Six weeks later the animals were sacrificed and HDV replication in normal liver tissues and in center masses of HCCs was evidenced by Northern analysis, real-time polymerase chain reaction assay, and immunohistochemistry. Based on accumulation levels of HDV RNAs and numbers of infected cells, the efficiency of wHDV infection appears to be comparable in most HCCs and normal liver tissues. CONCLUSION: Cells of WHV-induced HCCs are susceptible to HDV infection in vivo, and therefore express functional putative WHV receptors and support the steps of the attachment/entry governed by the hepadnavirus envelope proteins. Because others previously hypothesized that hepadnavirus-induced HCCs are resistant to reinfection with a hepadnavirus in vivo, our data suggest that if such a resistance exists it likely occurs via a block at the post-entry step. The demonstrated ability of HDV to infect already formed HCCs may facilitate development of novel strategies further dissecting the mechanism of liver pathogenesis associated with HDV infection.