i.v. administration of adenoviral vectors of more than 109 infectious units/mouse results in infection and Ag expression in more signaling pathway than 90% of hepatocytes and acute self-limiting viral hepatitis.[18, 19] In this study, to develop a useful animal model in the development of immunotherapy for chronic hepatitis C, we examined the responses
of intrahepatic CD8 T cells of HCV core transgenic (Tg) mice with various infectious doses of HCV-NS3-recombinant adenovirus (Ad-HCV-NS3). C57BL/6 MICE WERE purchased from Clea Japan (Tokyo, Japan), and Tokyo Laboratory Animal Science (Tokyo, Japan). Production of HCV core Tg mice has been described. The core gene of HCV placed downstream of a transcriptional regulatory region from hepatitis B virus, which has been shown to allow an expression of genes in Tg mice without interfering with mouse development, was introduced into C57BL/6 mouse embryos (Clea Japan). Eight- to 10-week-old mice were used for all experiments. The mice were housed in appropriate animal care facilities at Saitama Medical University (Saitama, Japan) and were handled according to international guidelines. The experimental protocols were approved by the Animal Research Committee of Saitama Medical University (#855). Adenovirus HCV-NS3 expressing the fusion protein, comprising the entire HCV-NS3
and 3X flag, was constructed by using the AdEasy XL adenoviral vector system (Agilent Technologies, Santa Clara, CA, USA). The HCV-NS3 gene corresponding to amino acid residues 1027–1657 was amplified from the plasmid pBRTM/HCV1-3011con which U0126 clinical trial contains
the entire DNA sequence derived from the HCV H77 clone (kindly provided by Charls M. Rice, The Rockefeller University, New York, NY, USA) by polymerase chain reaction. The recombinant Ad-HCV-NS3 vector was linearized by PacI digestion, and then transfected into 293 cells using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) to generate adenovirus. Ad-HCV-NS3 expressing transgene NS3 was amplified in 293 cells, purified by a series of cesium chloride SPTBN5 ultracentrifugation gradients and stored at −80°C until use. Mice were injected i.v. with 2 × 107, 1 × 109 and 1 × 1010 plaque-forming units (PFU) of Ad-HCV-NS3 or Adψ5 control vector. The experimental protocol regarding construction of recombinant adenovirus and infection of mice was approved by the Recombinant DNA Advisory Committee of Saitama Medical University (#1073). The liver was perfused with phosphate-buffered saline (PBS) plus 0.05% collagenase via the portal vein. Perfused livers were smashed through a 100-μm cell strainer (BD Biosciences, San Jose, CA, USA). The cell suspension was centrifuged with 35% Percoll at 320 g for 10 min, and the cell pellet was cultured in a plastic Petri dish in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS; R-10) for 1.5 h to remove adherent cells.