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.

Kinetics of viremia and acute liver injury in relation to outcome of neonatal woodchuck hepatitis virus infection.

The kinetics of serum viral responses and acute liver injury were studied during neonatal woodchuck hepatitis virus (WHV) infection in relation to the chronic or resolved outcome. The mean concentrations of serum WHV DNA and surface antigen were significantly higher by week 10 post infection in chronic infections compared to resolving infections, and diverged even further by the time of peak viral load development in serum (week 12). After week 12, these viral markers were detected less frequently with time and at lower concentrations in the resolved outcome. In both outcomes, mean serum activities of hepatic enzymes became increased significantly above baseline by weeks 10-12, peaked at week 14, and normalized by weeks 20-22, thus indicating transient acute liver injury. The increasing liver injury responses were comparable between outcomes at week 12, when serum viral load was markedly higher in the developing chronic infections. This suggested a deficiency in early non-cytolytic control of infection in the chronic outcome. At week 14, liver injury was significantly greater in the resolved outcome and associated with higher mean Fas ligand (FasL) and perforin messenger RNAs (mRNAs) in liver compared to the chronic outcome. This indicated greater immune-mediated killing of infected hepatocytes during resolution. Thus, chronicity as an outcome of neonatal WHV infection develops relatively early during the acute phase of infection due to reduced immune-mediated clearance of infected hepatocytes by both cytolytic and non-cytolytic processes.

Clevudine therapy with vaccine inhibits progression of chronic hepatitis and delays onset of hepatocellular carcinoma in chronic woodchuck hepatitis virus infection.

We examined a rational approach to therapy of chronic hepatitis B virus (HBV) infection that utilized the reduction of viral load combined with appropriately timed immune modulation/stimulation. In a placebo-controlled study, chronic woodchuck hepatitis virus (WHV) carrier woodchucks received clevudine (L-FMAU), previously shown to have especially potent and sustained antiviral activity in woodchucks, for 32 weeks followed by WHV surface antigen (WHsAg) alum-adjuvanted vaccine at 32, 36, 40 and 48 weeks. Clevudine induced significant reductions in viraemia, surface antigenaemia, hepatic WHV nucleic acids, and hepatic core and surface antigens. Viral replication markers remained markedly suppressed in 75% of the clevudine-treated woodchucks following drug withdrawal, but remained at high levels in the vaccine monotherapy and placebo groups. Combination drug and vaccine therapy had benefits based on sustained reduction of viraemia, antigenaemia, and hepatic WHV DNA and RNA; inhibition of progression of chronic hepatitis; reduced frequency of chronic liver injury; and delayed onset of hepatocellular carcinoma (HCC). Combination therapy contributed to prevention of HCC in up to 38% of treated carriers, although the growth rate of established HCC was not affected. This study demonstrates enhanced benefits of combination chemo-immunotherapy against viral load and disease progression in chronic hepadnaviral infection, and provides a platform for further development of such treatment regimens.

Lymphosarcoma in the laboratory woodchuck (Marmota monax).

From 1979 to 1999,28 cases of lymphosarcoma were identified in the Cornell University woodchuck colony (prevalence rate: 152/100,000/yr). The prevalence of lymphosarcoma was similar in woodchucks not infected with the woodchuck hepatitis virus (WHV) and in chronic carriers of WHV. Males (13) and females (15) alike were affected (mean +/- SD age 4.7 +/- 2.92 years; range, 0.5 to 9 years). On the basis of the major organ system involved, woodchuck lymphosarcoma was classified as multicentric (12 cases, 43%), alimentary (5 cases, 18%), cranial mediastinal (5 cases, 18%), and miscellaneous (6 cases, 21%). A cutaneous form was not observed. Morphologic criteria similar to those of the Kiel classification were used for light microscopic classification. All Kiel categories-except the immunoblastic form-were found: 17 cases (61%) were centroblastic, and 6 were lymphocytic (21%). Other categories (centrocytic and plasmacytoid) were recognized less frequently. Immunophenotyping of 27 cases revealed 15 (56%) B cell (CD3-/CD79a+ or CD3-/BLA.36+), 7 (26%) T cell (CD3+/CD79a-/BLA.36-), and 5 (18%) non-T non-B cell (CD3-CD79a-/BLA.36-) lymphosarcomas. Lymphosarcoma in woodchucks develops at a higher rate than that observed in humans or companion animals, and WHV infection has no effect on prevalence. The anatomic and Kiel classification used in domestic species also can be used in woodchucks. Commercially available alpha-CD3, alpha-CD79a, and alpha-BLA.36 antibodies were useful for immunophenotyping woodchuck lymphosarcomas.