Development of a GB virus B marmoset model and its validation with a novel series of hepatitis C virus NS3 protease inhibitors

Abstract

GB virus B (GBV-B), a flavivirus closely related to HCV, has previously been shown to infect and replicate to high titers in tamarins (Saguinus sp.). This study describes the use of GBV-B infection and replication in the common marmoset (Callithrix jacchus) for the successful development and validation of a surrogate animal model for hepatitis C virus (HCV). Infection of marmosets with GBV-B produced a viremia that peaked at 10(8) to 10(9) genome copies/ml for a period of 40 to 60 days followed by viral clearance at 60 to 80 days postinfection. Passage of the initial tamarin-derived GBV-B in marmosets produced an infectious stock that gave a more reproducible and consistent infection in the marmoset. Titration of the virus stocks in vivo indicated that they contained 1 infectious unit for every 1,000 genome copies. Cultures of primary marmoset hepatocytes were also successfully infected with GBV-B, with high levels of virus detected in supernatants and cells for up to 14 days postinfection. Treatment of GBV-B-infected hepatocyte cultures with a novel class of HCV protease inhibitor (pyrrolidine 5,5 trans-lactams) reduced viral levels by more than 2 logs. Treatment of GBV-B-infected marmosets with one such inhibitor resulted in a 3-log drop in serum viral titer over 4 days of therapy. These studies provide the first demonstration of the in vivo efficacy of a small-molecule inhibitor for HCV in an animal model and illustrate the utility of GBV-B as a surrogate animal model system for HCV.

FIG. 1.

Infection of tamarins with GBV-B infectious serum (tam-G17). GBV-B serum levels (measured using Taqman assays and indicated in ge per milliliter) (•) and liver damage results (measured according to serum levels of ALT [▪] and GLDH [□]) and indicated in international units per liter [IU/L]) are shown.

FIG. 2.

Comparison of replication profiles of two marmosets infected with GBV-B (tam-G17 infectious serum). (a) Marmoset Y037 results, a typical example of robust GBV-B replication. (b) Marmoset Y191 results, an example of poor GBV-B replication with delayed and low-level viremia. GBV-B serum levels (measured using Taqman assays and indicated in ge per milliliter) (•) and liver damage results (measured according to serum levels of ALT [▪] and GLDH [□]) and indicated in international units per liter [IU/L]) are shown.

FIG. 3.

Titration of GBV-B infectious sera (marm-Y243) from marmosets to determine infectious dose. GBV-B serum levels are shown for animals infected with the following amounts of virus: 500,000 ge (AA176) or 50,000 ge (Y350) (a), 5,000 ge (b), 500 ge (c), and 50 ge (d).

FIG. 4.

Rechallenge of marmosets previously infected with GBV-B. GBV-B serum levels were measured for animals rechallenged with 500,000 ge; the animals had initially received the following amounts of homologous virus stock as the primary infection: 500,000 ge (AA176) or 50,000 ge (Y350) (a), 5,000 ge (b), 500 ge (c), and 50 ge (d).

FIG. 5.

Replication of GBV-B in primary hepatocytes. Hepatocytes from a marmoset (a and b) or tamarin (c and d) were inoculated with either infectious (black bars) or UV-irradiated (grey-shaded bars) GBV-B (tam-G17 sera). Hepatocytes were cultured in SFM, and supernatants and cell pellets were harvested periodically for the next 14 days. (a and c) GBV-B RNA levels (quantified using Taqman assays) measured in culture supernatants and expressed as ge per milliliter of supernatant. Cultures had a total volume of 2 ml per well. (b and d) GBV-B RNA levels measured in cell pellets and expressed as ge per well (approximately 1 million cells per well). Error bars represent the standard deviations between duplicate cultures. The limit of detection in this experiment is represented by the dotted line. In a subsequent experiment, the GBV-B RNA level in cells on day 4 was measured as 1.7 × 10⁶ ge/well (★), which is more consistent with other data. nd, not done due to limitations of hepatocyte numbers.

FIG. 6.

Titration of tamarin- and marmoset-derived GBV-B stocks in marmoset hepatocyte cultures. Marmoset hepatocyte cultures were infected with either tam-G17 (black bars) or marm-Y243 (gray-shaded bars) in 10-fold dilutions from 10⁹ to 10⁵ ge per well. At 8 days p.i., GBV-B RNA levels in supernatants were measured using Taqman assays and are expressed as ge per milliliter. Error bars represent the standard deviations between duplicate cultures. nd, not done. The limit of detection in this experiment is represented by the dotted line.

FIG. 7.

Inhibition of GBV-B replication in primary marmoset hepatocytes following treatment with 5,5 pyrrolidine trans-lactams. Cultures of primary marmoset hepatocytes were inoculated with GBV-B (marm-Y243). After removal of the inoculum, cells were cultured in either SFM alone or SFM containing various concentrations of one of three trans-lactams. At 3 days later, supernatants were harvested; GBV-B RNA levels were measured using Taqman assays and are expressed as ge per milliter. Error bars represent the standard deviations between duplicate cultures.

FIG. 8.

Efficacy of trans-lactam GW0014X in the GBV-B marmoset model following treatment during the early phase of infection. Marmosets received one prophylactic subcutaneous dose of either GW0014X (30 mg/kg of body weight; n = 4) or vehicle (corn oil; n = 5) 4 h prior to infection with GBV-B marm-Y243 stock. Dosing then continued at twice a day for 7 days, as indicated by the black line at the top of the chart. Serum GBV-B RNA levels (measured using Taqman assays) were determined at days 4, 7, and 15 p.i. Marmosets that received GW0014X are represented individually by open symbols, while the average viral loads for the control marmosets (dosed with vehicle only) are represented by black circles. The error bars represent the standard deviations from the means for this group. The limit of detection for the Taqman assay is 5 × 10⁴ ge/ml and is represented by the dotted line.

FIG. 9.

Efficacy of trans-lactam GW0014X in the GBV-B marmoset model following treatment during the peak of virus replication. One marmoset (•) received 30 mg of GW0014X/kg of body weight, while two control marmosets (□ and Δ) received vehicle (corn oil). Compounds were given subcutaneously twice a day for 4 days between days 39 and 43 p.i., as indicated by the black line at the top of the chart. Serum GBV-B RNA levels (measured using Taqman assays) were determined on the day prior to therapy, in the middle of therapy, and then 1 day and 1 week after the completion of therapy. The limit of detection for the Taqman assay is 5 × 10⁴ ge/ml and is represented by the dotted line.