Inhibition of hepatitis B virus DNA replication by a thermostable interferon-gamma variant

Background: Treatment of HBV chronic carriers using interferon (IFN)-alpha or nucleoside/nucleotide analogues fails to suppress viral infection. Type-II IFN-gamma has been shown to inhibit HBV replication. The goal of the present work was to evaluate the antiviral efficacy against HBV of a thermostable IFN-gamma variant isolated using Massive Mutagenesis (R) and thermoresistant selection (THR (TM)) technologies. Methods: The thermostability of wild-type (wt) and S63C IFN-gamma was determined in vitro and in vivo. Activation of the IFN-gamma responsive element by wt and S63C IFN-gamma was tested using a luciferase assay. HepG2.2.15 cells constitutively expressing HBV were used to analyse the antiviral activity of wt and S63C IFN-gamma against HBV replication. Intracellular HBV DNA was detected by Southern blot and quantified by real-time PCR analyses. Results: S63C IFN-gamma was shown to be more thermostable and had a longer half-life than wt IFN-gamma. Both wt and 563C IFN-gamma displayed a similar capacity to activate the IFN pathway. The treatment of HepG2.2.15 cells with wt or S63C IFN-gamma induced the inhibition of HBV viral replication. After heating, S63C IFN-gamma displayed better conservation of its antiviral activity against HBV when compared with wt IFN-gamma. Conclusions: These results confirm that the THR (TM) method can be used to isolate mutants with enhanced thermostability and demonstrate that a thermostable IFN-gamma variant presents antiviral properties against HBV replication. This molecule could provide a new strategy to treat patients who do not respond to antiviral therapy.