As shown Fig. 4C, two out of three woodchucks treated with CTX responded with wIFN-γ production after IL-12 in vitro stimulation. Viral load was analyzed 30 and 10 days before and then again at 1, 4, 10, 30, 50, 70, and 90 days after CTX treatment and no antiviral effect was observed (Fig. 4D). After demonstrating that TGF-β1 inhibition and Treg depletion are able to restore the responsiveness to IL-12 in chronic WHV carriers with high viral load, we proceeded to treat these animals with a gene therapy vector encoding IL-12, alone or in combination with P17 or CTX administration. BAY 80-6946 research buy A single dose of 1 × 1011 infectious units of HC-Ad/RUmIL-1218, 19 (a high-capacity adenoviral vector that express IL-12
under the control of a liver-specific, mifepristone-inducible promoter) was administered during laparotomy by intrahepatic injection into three different liver areas. Three woodchucks received the vector alone, three other animals received the vector in combination with P17 peptide, three other animals received the vector in combination with a low dose of CTX, and four other animals were left untreated and served as controls. A schematic representation of the experimental design is provided in Fig. 5A. IL-12 expression was induced daily for 40 days by intraperitoneal administration of 500 μg/kg mifepristone, starting 40 days after vector administration. The P17 peptide was administered
in 10 doses of 5 mg/kg given every other day and starting 30 days selleck chemicals llc prior to IL-12 induction, P17 treatment and IL-12 induction was separated because the recovery of lymphocyte response to IL-12 stimulation only occurs after a considerable
time lag following P17 administration, as demonstrated for the majority of woodchucks that showed maximum IFN-γ production at day 52 following the initial dose of P17 peptide (Fig. 3C). CTX was administered at a single low dose of 20 mg/kg and IL-12 induction learn more was initiated 10 days later, when FoxP3 expression was undetectable in the liver of CTX-treated animals, as shown in Fig. 4B. Liver biopsies were obtained prior to vector injection during laparotomy, and then again 20 days after the completion of the induction cycle. Following mifepristone administration, IL-12 expression was highly variable in individual woodchucks but was comparable between the three experimental groups (Fig. 5B). Viremia in serum was analyzed weekly but no changes in viral load were detected (Fig. 5C). Notably, the analysis of hepatic expression of immunosuppressive molecules revealed an increased tolerogenic environment of the liver. Specifically, an increase in FoxP3 expression was observed in all animals treated with IL-12 (Fig. 6A). This finding was confirmed by immunohistochemical staining with an anti-FoxP3 antibody (Fig. 6B). A significant increase in PD-1 mRNA expression was also observed in the majority of animals (Fig. 6A).