The antitumoral efficacy of a SFV vector expressing high levels of IL-12 (SFV-enhIL-12) was investigated in six woodchucks with established chronic WHV infection and primary HCC. The results demonstrate that SFV-delivered IL-12 expression nearly produced a dose-dependent, partial tumor remission that was associated with a general activation of cellular immune responses against HCC. The antitumoral activity, in addition to an antiviral activity against WHV, and the favorable safety profile in woodchucks suggest that a therapeutic approach based on SFV-enhIL-12 may represent a treatment strategy for HCC in patients with chronic HBV infection, but the overall results also indicate that this approach needs further improvement for inducing a complete tumor remission. MATERIALS AND METHODS Cell lines and animals.

The BHK-21 cell line (ATCC CCL-10) was cultured in Glasgow minimum essential medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (Invitrogen) as described previously (39). The woodchuck HCC-derived cell line WCH17 (ATCC CRL-2082) and the human HCC-derived cell lines HepB3 (ATCC HB-8064) and Huh7 (our laboratory stock) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). All experimental procedures involving woodchucks were performed under protocols approved by the Cornell University Institutional Animal Care and Use Committee. Woodchucks were born to WHV-negative females and reared in environmentally controlled laboratory animal facilities at Cornell University.

Woodchucks were inoculated at 3 days of age by subcutaneous inoculation with 5 �� 106 50% woodchuck infectious doses of a standardized WHV inoculum (cWHV7P2) (11). Persistence of WHV infection was based on the consecutive detection of WHV DNA and WHV surface antigen (WHsAg) in serum from 3 months of age. All chronic WHV carrier woodchucks had developed HCC at the beginning of the study as determined by hepatic ultrasound examination and elevated serum of ��-glutamyl-transferase (GGT) activity (��11 IU/liter, compared to 3 to 4 IU/liter in chronic WHV carrier woodchucks without HCC). Cell transfection and virus production. Plasmids pSFV-Luc and pSFV-enhIL-12 and the generation of recombinant SFV vp in BHK-21 cells have been described previously (39). Plasmid pSFV3-LacZ was kindly provided by P.

Liljestr?m (Karolinska Institute, Stockholm, Sweden) (24). RNA synthesis from SFV plasmids, transfection into BHK-21 cells by electroporation, and packaging of recombinant RNA into SFV vp were performed as described previously (39, 43). Briefly, BHK-21 cells were coelectroporated with the recombinant RNA, Cilengitide together with two helper RNAs (i.e., SFV-helper-C-S219A and SFV-helper-S2 RNAs), which provided in trans the capsid and the spike proteins. SFV vp were harvested and purified by ultracentrifugation as described previously (15).