Christine A. Bellezza

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.

Antiviral Activities of Oral 1-O-Hexadecylpropanediol-3-Phosphoacyclovir and Acyclovir in Woodchucks with Chronic Woodchuck Hepatitis Virus Infection

ABSTRACT Acyclovir triphosphate is a potent inhibitor of hepatitis B virus DNA polymerase, but acyclovir treatment provides no benefit in patients with hepatitis B virus infection. This is due in part to the fact that hepatitis B virus, unlike herpes simplex virus, does not code for a viral thymidine kinase which catalyzes the initial phosphorylation of acyclovir. We synthesized 1-O-octadecyl-sn-glycero-3-phospho (3-P)-acyclovir and found that it was highly active in reducing hepatitis B virus replication in 2.2.15 cells, while acyclovir was inactive. The greater antiviral activity of 1-O-octadecyl-sn-glycero-3-P-acyclovir appeared to be due to liver cell metabolism of the compound to acyclovir monophosphate (K. Y. Hostetler et al., Biochem. Pharmacol. 53:1815–1822, 1997). However, a closely related compound without a hydroxyl group at the sn-2 position of glycerol, 1-O-hexadecylpropanediol-3-P-acyclovir, was more active and selective in 2.2.15 cells in vitro. In this study, we treated woodchucks chronically infected with woodchuck hepatitis virus with increasing oral doses of 1-O-hexadecylpropanediol-3-P-acyclovir and assessed the response to therapy versus acyclovir or a placebo. At a dosage of 10 mg/kg of body weight twice a day, the test compound significantly inhibited viral replication in vivo, as indicated by a 95% reduction in serum woodchuck hepatitis virus DNA levels and by a 54% reduction in levels of woodchuck hepatitis virus replicative intermediates in the liver. Higher doses were somewhat less effective. In contrast, 20 mg of acyclovir/kg twice daily, a 5.3-fold-higher molar dosage, had no demonstrable activity against woodchuck hepatitis virus. Oral 1-O-hexadecylpropanediol-3-P-acyclovir appeared to be safe and effective in chronic woodchuck hepatitis virus infection.

Semliki Forest Virus Expressing Interleukin-12 Induces Antiviral and Antitumoral Responses in Woodchucks with Chronic Viral Hepatitis and Hepatocellular Carcinoma

ABSTRACT A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with ≥1.5-log10 reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.

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

ABSTRACT 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.

Interferon-gamma-associated responses to woodchuck hepatitis virus infection in neonatal woodchucks and virus-infected hepatocytes.

Summary.  Acute hepatitis and recovery from woodchuck hepatitis virus (WHV) infection involves increased intrahepatic expression of interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) mRNAs. In the present study, recovery correlated with increased intrahepatic expression of mRNAs for major histocompatibility complex class 1 (MHC1), β2‐microglobulin, 2′5′‐oligoadenylate synthetase (2′5′‐OAS), and indoleamine dioxygenase (IDO). By comparison, acute WHV infection progressing to chronicity was associated with diminished expression of these IFN‐γ‐associated mRNAs in liver. Transfection of WHV‐infected primary hepatocytes (WPH) from WHV carriers with an IFN‐γ‐expressing plasmid (pIFN‐γ) resulted in dose‐dependent accumulations of MHC1, TNF‐α, 2′5′‐OAS, and IDO mRNAs within 96 h. Markers of T cells and immune‐mediated cytotoxicity that accumulate in recovering liver were not apparent in WPH based on the relative lack of CD3, CD4, Fas ligand, perforin, and granzyme B mRNAs. Expression of pIFN‐γ, and TNF‐α‐expressing plasmid (pTNF‐α), did not affect total WHV RNA, or fully double‐stranded WHV DNA in WPH, but each reduced some of the replicative intermediate (RI) species of WHV DNA synthesis. WPH treated with recombinant IFN‐α protein had a higher fold induction of 2′5′‐OAS mRNA associated with partial reductions in WHV RNAs and the major RI species. Thus, IFN‐γ expression in carrier WPH induced several host responses often observed in liver of recovering woodchucks, and impaired a stage of WHV DNA synthesis by a non‐cytolytic mechanism mediated by TNF‐α. Local enhancement of IFN‐γ‐associated responses in chronic WHV‐infected hepatocytes may promote therapeutic antiviral effects, but additional effector mechanisms evident during recovery appear necessary for more complete clearance of WHV infection.