Woodchuck Hepatitis Virus Surface Antigen Research Articles

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

Background and AimsGS‐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 ResultsWHV‐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.ConclusionsFinite, 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.

Efficacy of an Inhibitor of Hepatitis B Virus Expression in Combination With Entecavir and Interferon-α in Woodchucks Chronically Infected With Woodchuck Hepatitis Virus.

RG7834 is a small‐molecule inhibitor of hepatitis B virus (HBV) gene expression that significantly reduces the levels of hepatitis B surface antigen (HBsAg) and HBV DNA in a humanized liver HBV mouse model. In the current study, we evaluated the potency of RG7834 in the woodchuck model of chronic HBV infection, alone and in combination with entecavir (ETV) and/or woodchuck interferon‐α (wIFN‐α). RG7834 reduced woodchuck hepatitis virus (WHV) surface antigen (WHsAg) by a mean of 2.57 log10 from baseline and WHV DNA by a mean of 1.71 log10. ETV + wIFN‐α reduced WHsAg and WHV DNA by means of 2.40 log10 and 6.70 log10, respectively. The combination of RG7834, ETV, and wIFN‐α profoundly reduced WHsAg and WHV DNA levels by 5.00 log10 and 7.46 log10, respectively. However, both viral parameters rebounded to baseline after treatment was stopped and no antibody response against WHsAg was observed. Effects on viral RNAs were mainly seen with the triple combination treatment, reducing both pregenomic RNA (pgRNA) and WHsAg RNA, whereas RG7834 mainly reduced WHsAg RNA and ETV mainly affected pgRNA. When WHsAg was reduced by the triple combination, peripheral blood mononuclear cells (PBMCs) proliferated significantly in response to viral antigens, but the cellular response was diminished after WHsAg returned to baseline levels during the off‐treatment period. Consistent with this, Pearson correlation revealed a strong negative correlation between WHsAg levels and PBMC proliferation in response to peptides covering the entire WHsAg and WHV nucleocapsid antigen. Conclusion: A fast and robust reduction of WHsAg by combination therapy reduced WHV‐specific immune dysfunction in the periphery. However, the magnitude and/or duration of the induced cellular response were not sufficient to achieve a sustained antiviral response.

Chinese woodchucks with different susceptibility to WHV infection differ in their genetic background exemplified by cytochrome B and MHC-DRB molecules

BackgroundChinese woodchucks (M. himalayana) were recently found to be susceptible to woodchuck hepatitis virus (WHV) infection. In this study, we aimed to determine the susceptibility to WHV infection of M. himalayana from different areas and their association with the animal genetic background exemplified by cytochrome B and MHC-DRB molecules.MethodsAnimals from four different areas in Qinghai province were inoculated with WHV59 strains. The virological markers including WHV surface antigen (WHsAg), WHV core antibody (WHcAb), and WHV DNA in serum were measured by ELISA and Real-time PCR, respectively. The sequences of cytochrome B gene and MHC-DRB molecules were obtained and sorted with Clustalx software. The nucleotide variation sites were identified using MEGA5 software.ResultsThe animals from four different areas had different susceptibility to WHV infection. Animals from TR and TD areas had a high level of long-lasting viremia, while those from GD and WL areas had a low level of transient viremia after WHV inoculation. All of the animals belong to the same subspecies M. himalayana robusta identified by cytochrome B gene sequences. Based on their nucleotide variation pattern, 8 alleles of cytochrome B gene were identified, and 7 MHC-DRB alleles were identified. Allele A of cytochrome B and Allele Mamo-DRB1*02 of MHC-DRB was found to be frequent in animals from TR and TD areas, while Allele H of cytochrome B and Allele Mamo-DRB1*07 of MHC-DRB was predominant in animals from GD and WL areas.ConclusionChinese woodchucks from different areas differed in their susceptibility to WHV infection, though they belong to the same subspecies M. himalayana robusta. The genetic background exemplified by cytochrome B and MHC-DRB differed in Chinese woodchucks with different susceptibility to WHV infection.

Safety and efficacy of anti-PD-L1 therapy in the woodchuck model of HBV infection.

Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters. We used woodchucks infected with WHV to assess the safety and efficacy of anti-PD-L1 monoclonal antibody therapy (αPD-L1) in a variety of WHV infection states. Experimentally-infected animals lacked PD-1 or PD-L1 upregulation compared to uninfected controls, and accordingly, αPD-L1 treatment in lab-infected animals had limited antiviral effects. In contrast, animals with naturally acquired WHV infections displayed elevated PD-1 and PD-L1. In these same animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone, but with efficacy restricted to a minority of animals. Pre-treatment WHV surface antigen (sAg) level was identified as a statistically significant predictor of treatment response, while PD-1 expression on peripheral CD8+ T cells, T cell production of interferon gamma (IFN-γ) upon in vitro antigen stimulation (WHV ELISPOT), and circulating levels of liver enzymes were not. To further assess the safety of this strategy, αPD-L1 was tested in acute WHV infection to model the risk of liver damage when the extent of hepatic infection and antiviral immune responses were expected to be the greatest. No significant increase in serum markers of hepatic injury was observed over those in infected, untreated control animals. These data support a positive benefit/risk assessment for blockade of the PD-1:PD-L1 pathway in CHB patients and may help to identify patient groups most likely to benefit from treatment. Furthermore, the efficacy of αPD-L1 in only a minority of animals, as observed here, suggests that additional agents may be needed to achieve a more robust and consistent response leading to full sAg loss and durable responses through anti-sAg antibody seroconversion.

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

BackgroundApproximately 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.MethodsIn 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.ResultsIt 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.ConclusionsAlthough 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.

Antiviral Efficacy and Host Immune Response Induction during Sequential Treatment with SB 9200 Followed by Entecavir in Woodchucks.

SB 9200, an orally bioavailable dinucleotide, activates the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) causing the induction of the interferon (IFN) signaling cascade for antiviral defense. The present study evaluated the overall antiviral response in woodchucks upon induction of immune response, first with SB 9200 followed by Entecavir (ETV) versus reduction of viral burden with ETV followed by SB 9200 immunomodulation. Woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated orally with SB 9200 (30 mg/kg/day) and ETV (0.5 mg/kg/day). Group 1 received ETV for 4 weeks followed by SB 9200 for 12 weeks. Group 2 received SB 9200 for 12 weeks followed by ETV for 4 weeks. At the end of treatment in Group 2, average reductions of 6.4 log10 in serum WHV DNA and 3.3 log10 in WHV surface antigen were observed whereas in Group 1, average reductions of 4.2 log10 and 1.1 log10 in viremia and antigenemia were noted. Both groups demonstrated marked reductions in hepatic WHV nucleic acid levels which were more pronounced in Group 2. Following treatment cessation and the 8-week follow-up, recrudescence of viral replication was observed in Group 1 while viral relapse in Group 2 was significantly delayed. The antiviral effects observed in both groups were associated with temporally different induction of IFN-α, IFN-β, and IFN-stimulated genes in blood and liver. These results suggest that the induction of host immune responses by pretreatment with SB 9200 followed by ETV resulted in antiviral efficacy that was superior to that obtained using the strategy of viral reduction with ETV followed by immunomodulation.

Antiviral Efficacy and Host Innate Immunity Associated with SB 9200 Treatment in the Woodchuck Model of Chronic Hepatitis B.

SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-β and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway.

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

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. 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.

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

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). 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. 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. 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.

Primary seronegative but molecularly evident hepadnaviral infection engages liver and induces hepatocarcinoma in the woodchuck model of hepatitis B.

Hepadnavirus at very low doses establishes in woodchucks asymptomatic, serologically undetectable but molecularly evident persistent infection. This primary occult infection (POI) preferentially engages the immune system and initiates virus-specific T cell response in the absence of antiviral antibody induction. The current study aimed to determine whether POI with time may culminate in serologically identifiable infection and hepatitis, and what are, if any, its pathological consequences. Juvenile woodchucks were intravenously injected with inocula containing 10 or 100 virions of woodchuck hepatitis virus (WHV) to induce POI and followed for life or up to 5.5 years thereafter. All 10 animals established molecularly detectable infection with virus DNA in serum (<100–200 copies/mL) and in circulating lymphoid cells, but serum WHV surface antigen and antibodies to WHV core antigen remained undetectable for life. By approximately 2.5–3.5 years post-infection, circulating virus transiently increased to 103 copies/mL and virus replication became detectable in the livers, but serological markers of infection and biochemical or histological evidence of hepatitis remained undetectable. Nonetheless, typical hepatocellular carcinoma (HCC) developed in 2/10 animals. WHV DNA integration into hepatic and lymphatic system genomes was identified in 9/10 animals. Virus recovered from the liver virus-negative or virus-positive phases of POI displayed the wild-type sequence and transmitted infection to healthy woodchucks causing hepatitis and HCC. In summary, for the first time, our data demonstrate that an asymptomatic hepadnaviral persistence initiated by very small amounts of otherwise pathogenic virus, advancing in the absence of traditional serological markers of infection and hepatitis, coincides with virus DNA integration into the host's hepatic and immune system genomes, retains liver pro-oncogenic potency and is capable of transmitting liver pathogenic infection. This emphasizes the role for primary occult hepatitis B virus infection in the development of seemingly cyptogenic HCC in seronegative but virus DNA reactive patients.

Electroporation Enhances Immunogenicity of a DNA Vaccine Expressing Woodchuck Hepatitis Virus Surface Antigen in Woodchucks

The development of therapeutic vaccines for chronic hepatitis B virus (HBV) infection has been hampered by host immune tolerance and the generally low magnitude and inconsistent immune responses to conventional vaccines and proposed new delivery methods. Electroporation (EP) for plasmid DNA (pDNA) vaccine delivery has demonstrated the enhanced immunogenicity of HBV antigens in various animal models. In the present study, the efficiency of the EP-based delivery of pDNA expressing various reporter genes first was evaluated in normal woodchucks, and then the immunogenicity of an analog woodchuck hepatitis virus (WHV) surface antigen (WHsAg) pDNA vaccine was studied in this model. The expression of reporter genes was greatly increased when the cellular uptake of pDNA was facilitated by EP. The EP of WHsAg-pDNA resulted in enhanced, dose-dependent antibody and T-cell responses to WHsAg compared to those of the conventional hypodermic needle injection of WHsAg-pDNA. Although subunit WHsAg protein vaccine elicited higher antibody titers than the DNA vaccine delivered with EP, T-cell response rates were comparable. However, in WHsAg-stimulated mononuclear cell cultures, the mRNA expression of CD4 and CD8 leukocyte surface markers and Th1 cytokines was more frequent and was skewed following DNA vaccination compared to that of protein immunization. Thus, the EP-based vaccination of normal woodchucks with pDNA-WHsAg induced a skew in the Th1/Th2 balance toward Th1 immune responses, which may be considered more appropriate for approaches involving therapeutic vaccines to treat chronic HBV infection.

Glucosidase inhibition enhances presentation of de-N-glycosylated hepatitis B virus epitopes by major histocompatibility complex class I in vitro and in woodchucks.

In this report, the possibility of pharmacologically altering the hepatitis B virus (HBV) epitopes presented by major histocompatibility complex class I on infected cells is demonstrated. The HBV middle envelope glycoprotein (MHBs) maturation appears to require calnexin-mediated folding. This interaction is dependent on glucosidases in the endoplasmic reticulum. Prevention of HBV envelope protein maturation in cultured cells through use of glucosidase inhibitors, such as 6-O-butanoyl castanospermine and N-nonyl deoxynorjirimycin, resulted in MHBs degradation by proteasomes. The de-N-glycosylation associated with polypeptide degradation was predicted to result in conversion of asparagine residues into aspartic acid residues. This prediction was confirmed by showing that peptides corresponding to the N-glycosylation sequons of MHBs, but with aspartic acid replacing asparagine, (1) can prime human cytotoxic T lymphocytes that recognize HBV-producing cells and (2) that the presentation of these envelope motifs by major histocompatibility complex class I is enhanced by incubation with glucosidase inhibitors. Moreover, although peripheral blood mononuclear cells isolated from woodchucks chronically infected with woodchuck hepatitis virus and vaccinated with woodchuck hepatitis virus surface antigen could be induced to recognize the natural MHBs asparagine-containing peptides, only cells isolated from animals treated with glucosidase inhibitor recognized the aspartic acid-containing peptides. These data suggest that pharmacological intervention with glucosidase inhibitors can alter the MHBs epitopes presented. This editing of the amino acid sequence of the polypeptide results in a new epitope, or "editope", with possible medical significance.

Identification of CD4+ and CD8+ T cell epitopes of woodchuck hepatitis virus core and surface antigens in BALB/c mice

A therapeutic vaccine against chronic hepatitis B virus (HBV) infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV infection and represent the best animal model for this hepadnavirus-induced disease. Using the BIMAS “HLA Peptide Binding Predictions” program, we have identified and further characterized novel H-2d-restricted CD8+ epitopes within the WHV core (peptides C#12–21, C#18–32, C#19–27, C#61–69) and surface antigens (peptides preS2#10–18, preS2#27–35, S#76–84, S#133–140 and S#257–265), respectively. These peptides bind to H-2d with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-γ producing T cells. More importantly, WHV core peptides C#19–27 and C#61–69 and WHV surface peptides S#133–140 and S#257–265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18–32, S#76–84, and S#257–265 resulted in significant production of IFN-γ. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against hepadnavirus infection.

Rapid immunity to vaccination with woodchuck hepatitis virus surface antigen using cationic liposome–DNA complexes as adjuvant

Complexes of cationic liposomes and non-coding DNA (CLDC) have shown promise as vaccine adjuvant. Using the woodchuck animal model of hepatitis B virus (HBV) infection, the immunogenic effects of CLDC were evaluated following vaccination with three doses of woodchuck hepatitis virus surface antigen (WHsAg) adjuvanted with either CLDC or conventional alum and administered intramuscularly (im) or subcutaneously (sc). IM vaccination with WHsAg and CLDC elicited antibodies earlier, in more woodchucks, and with higher titers than WHsAg and alum. After two vaccine doses, antibody titers were higher following im than sc administration. Woodchucks administered two vaccine doses sc received the third vaccine dose im, and antibody responses reached titers comparable to those elicited by im administration. Following the second vaccine dose, im vaccination with WHsAg and CLDC induced T cell responses to WHsAg and selected WHs peptides and expression of the leukocyte surface marker CD8 and of the Th1 cytokines interferon-gamma and tumor necrosis factor alpha in woodchucks. T cell responses and CD8/cytokine expression were diminished in woodchucks from the other groups suggesting that this vaccine regimen induced a skew toward Th1 immune responses. The present study in woodchucks demonstrates that CLDC-adjuvanted WHsAg vaccine administered im resulted in a more rapid induction of humoral and cellular immune responses compared to conventional, alum-adjuvanted WHsAg vaccine. While less rapid, the immune responses following sc administration can prime the im immune responses. This adjuvant activity of CLDC over alum may be beneficial for therapeutic vaccination in chronic HBV infection.

Antiviral Effects of Lamivudine, Emtricitabine, Adefovir Dipivoxil, and Tenofovir Disoproxil Fumarate Administered Orally Alone and in Combination to Woodchucks with Chronic Woodchuck Hepatitis Virus Infection

Adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate (TDF) are nucleotide analogs that inhibit the replication of wild-type hepatitis B virus (HBV) and lamivudine (3TC)-resistant virus in HBV-infected patients, including those who are coinfected with human immunodeficiency virus. The combination of ADV or TDF with other nucleoside analogs is a proposed strategy for managing antiviral drug resistance during the treatment of chronic HBV infection. The antiviral effect of oral ADV or TDF, alone or in combination with 3TC or emtricitabine (FTC), against chronic woodchuck hepatitis virus (WHV) infection was evaluated in a placebo-controlled study in the woodchuck, an established and predictive model for antiviral therapy. Once-daily treatment for 48 weeks with ADV plus 3TC or TDF plus FTC significantly reduced serum WHV viremia levels from the pretreatment level by 6.2 log(10) and 6.1 log(10) genome equivalents/ml serum, respectively, followed by TDF plus 3TC (5.6 log(10) genome equivalents/ml), ADV alone (4.8 log(10) genome equivalents/ml), ADV plus FTC (one survivor) (4.4 log(10) genome equivalents/ml), TDF alone (2.9 log(10) genome equivalents/ml), 3TC alone (2.7 log(10) genome equivalents/ml), and FTC alone (2.0 log(10) genome equivalents/ml). Individual woodchucks across all treatment groups also demonstrated pronounced declines in serum WHV surface antigen, characteristically accompanied by declines in hepatic WHV replication and the hepatic expression of WHV antigens. Most woodchucks had prompt recrudescence of WHV replication after drug withdrawal, but individual woodchucks across treatment groups had sustained effects. No signs of toxicity were observed for any of the drugs or drug combinations administered. In conclusion, the oral administration of 3TC, FTC, ADV, and TDF alone and in combination was safe and effective in the woodchuck model of HBV infection.

Immunogenicity in mice and rabbits of DNA vaccines expressing woodchuck hepatitis virus antigens

The licensed vaccine against hepatitis B virus (HBV) is an effective means to prevent infection, but is not an effective therapeutic strategy to treat established chronic infections when used alone. In an animal model of chronic HBV infection (the woodchuck experimentally infected with woodchuck hepatitis virus (WHV)), the combination of conventional vaccine and potent antiviral drugs has shown promise as a potential therapeutic intervention. This approach might be improved further through the application of newer vaccine technologies. In the present study, we evaluated electroporation (EP)-based intramuscular (i.m.) delivery of a codon-optimized DNA vaccine for the WHV surface antigen (WHsAg) in mice and rabbits. In mice, this immunization procedure compared favorably to vaccination by i.m. injection of the DNA vaccine or i.m. administration of a recombinant WHsAg–alum vaccine, exhibiting characteristics expected to be beneficial for a therapeutic vaccine strategy. These included dose efficiency, consistency, vigorous induction of antibody responses to WHsAg, as well as a Th1 bias. Following scale-up to rabbits, a species that approximates the size of the woodchuck, the EP dosing regimen was markedly more effective than conventional i.m. injection of the DNA vaccine. Taken together, these results provide the foundation for studies of EP-based DNA immunization in the woodchuck in order to further assess its potential as an immunotherapeutic approach for treatment of chronic HBV infection in humans.

Serologic Survey of Woodchuck Hepatitis Virus in North Carolina Woodchucks (Marmota monax)

The prevalence of woodchuck hepatitis virus (WHV) in wild populations of woodchucks is understudied and therefore unclear. Although infection is common in the southeastern region of Pennsylvania and surrounding states, it is virtually absent in New York and New England. Sera were collected from wild woodchucks from Orange County, North Carolina and tested for the presence of markers of current or previous infection with WHV. Of the 24 woodchucks tested, there were three animals (12.5%) with WHV surface antigen as well as antibodies to woodchuck hepatitis core antigen in their serum, indicative of active infection. There were four (17%) animals with antibodies to WHV core antigen but no woodchuck hepatitis surface antigen, indicative of prior infections. The remaining 17 animals had no detectable markers of WHV infection. These data indicate that WHV is present in central North Carolina at rates approaching those seen in endemic areas, such as the mid-Atlantic region of the United States.

Antiviral Effect of Orally Administered (−)-β-d-2-Aminopurine Dioxolane in Woodchucks with Chronic Woodchuck Hepatitis Virus Infection

(-)-beta-D-2-Aminopurine dioxolane (APD) is a nucleoside prodrug that is efficiently converted to 9-(beta-D-1,3-dioxolan-4-yl)guanine (DXG). DXG has antiviral activity in vitro against hepatitis B virus (HBV) but limited aqueous solubility, making it difficult to administer orally to HBV-infected individuals. APD is more water soluble than DXG and represents a promising prodrug for the delivery of DXG. A placebo-controlled, dose-ranging efficacy and pharmacokinetic study was conducted with woodchucks that were chronically infected with woodchuck hepatitis virus (WHV). APD was efficiently converted to DXG after oral and intravenous administrations of APD, with serum concentrations of DXG being higher following oral administration than following intravenous administration, suggestive of a considerable first-pass intestinal and/or hepatic metabolism. APD administered orally at 1, 3, 10, and 30 mg/kg of body weight per day for 4 weeks produced a dose-dependent antiviral response. Doses of 3 and 10 mg/kg/day reduced serum WHV viremia by 0.4 and 0.7 log(10) copies/ml, respectively. The 30-mg/kg/day dose resulted in a more pronounced, statistically significant decline in serum WHV viremia of 1.9 log(10) copies/ml and was associated with a 1.5-fold reduction in hepatic WHV DNA. Individual woodchucks within the highest APD dose group that had declines in serum WHV surface antigen levels, WHV viremia, and hepatic WHV DNA also had reductions in hepatic WHV RNA. There was a prompt recrudescence of WHV viremia following drug withdrawal. Therefore, oral administration of APD for 4 weeks was safe in the woodchuck model of chronic HBV infection, and the effect on serum WHV viremia was dose dependent.

Immunomodulation as an option for the treatment of chronic hepatitis B virus infection: preclinical studies in the woodchuck model

New therapeutic approaches for chronic hepatitis B virus infection based on immunomodulation are now under investigation. The woodchuck model for hepatitis B virus infection has emerged as a useful animal model for the evaluation of such approaches, after developing necessary assays and reagents for immunologic studies in this model. Conventional and novel vaccines such as DNA vaccines were tested in woodchucks for their ability to induce protective immune responses against challenge infection with the woodchuck hepatitis virus (WHV). Furthermore, immunotherapeutic approaches for the control of chronic hepadnaviral infection were evaluated in woodchucks. Immunizations with WHV proteins and DNA vaccines led to the development of antibodies to the WHV surface antigen and to a significant decrease of viral load in chronically WHV-infected woodchucks. Viral vector-mediated gene transfer was explored for the delivery of antiviral cytokines IFN-α in woodchucks and resulted in the decrease of viral replication. It is now generally accepted that a combination of antiviral treatment and immunization will be necessary to achieve successful immunomodulation with a long-term control of chronic hepatitis B virus infection.

Acute Resolving Woodchuck Hepatitis Virus (WHV) Infection Is Associated with a Strong Cytotoxic T-Lymphocyte Response to a Single WHV Core Peptide

Woodchucks infected with woodchuck hepatitis virus (WHV) are an excellent model for studying acute, self-limited and chronic hepadnaviral infections. Defects in the immunological response leading to chronicity are still unknown. Specific T-helper cell responses to WHV core and surface antigens (WHcAg and WHsAg, respectively) are associated with acute resolving infection; however, they are undetectable in chronic infection. Up to now, cytotoxic T-lymphocyte (CTL) responses could not be determined in the woodchuck. In the present study, we detected virus-specific CTL responses by a CD107a degranulation assay. The splenocytes of woodchucks in the postacute phase of WHV infection (18 months postinfection) were isolated and stimulated with overlapping peptides covering the whole WHcAg. After 6 days, the cells were restimulated and stained for CD3 and CD107a. One peptide (c96-110) turned out to be accountable for T-cell expansion and CD107a staining. Later, we applied the optimized degranulation assay to study the kinetics of the T-cell response in acute WHV infection. We found a vigorous T-cell response against peptide c96-110 with peripheral blood cells beginning at the peak of viral load (week 5) and lasting up to 15 weeks postinfection. In contrast, there was no T-cell response against peptide c96-110 detectable in chronically WHV-infected animals. Thus, with this newly established flow cytometric degranulation assay, we detected for the first time virus-specific CTLs and determined one immunodominant epitope of WHcAg in the woodchuck.