Study Of A Recombinant Adenovirus Vector Vaccine Expressing The NS3 Antigen Of HCV

Objective Because of hepatitis C virus (HCV) different gene types and the rate of high gene mutation of HCV, HCV vaccine development is now faced with a lot difficulties by using traditional vaccine development methods. The recombinant adenovirus vector can induce a high level of expression of the recombinant proteins and anti-viral specific immune responses. With this aim, we have constructed a replication-deficient recombinant adenovirus expressing HCV-NS3 protein (RAd-NS3) and evaluate its immunization effect in BALB/c mice.Methods The HCV-NS3 gene encoding for 302-935aa was amplified from plasmid pRc/NS3 by PCR and cloned into the transfer vector pAdTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E.coli strain BJ5183. Taking the the advantage of the high efficient homologous recombinant machinerypresented in bacteria, the recombinant adenoviral backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant adenovirus was propagated in 293 cells with high titers. We have also constructed the control recombinant adenovirus Ad-Track. The transcription of NS3 and expression of NS3 protein were detected by RT-PCR and Western blot in HepG2 cells infected with RAd-NS3. Six-week-old female BALB/c mice were inoculated intraperitioneally with 108 pfu of RAd-NS3 and Ad-Track each on weeks 0, 3, 5 and 7. Anti-HCV-NS3 antibodies were tested by ELISA. Cytotoxic T lymphocyte (CTL) activity to HCV antigen were detected by a standard 4-hr 51Cr release assays.Results we have constructed the recombinant adenoviral vector expressing NS3 antigen of HCV (RAd-NS3) and have demonstrated tumor cells infected with recombinant adenovirus vector RAd-NS3 can express NS3 antigen. Balb/c mice immunized with recombinant adenovirus RAd-NS3 can induce both anti-HCV humoral and T cytotoxic response. The highest titers of serum anti-HCV-NS3 were 1:10000 and antibody level average remains 1:1000 within 10weeks. CTL cytotoxic activity was higher in mice immunized with RAd-NS3 than in control mice. CTLs might induce approximately 53.22% lysis against SP2/0-NS3 target cells expressing NS3 antigen of HCV. No clear side effect was observed after immunization.Conclusions The recombinant adenoviral vector can stimulate specific humoral and celluler immune response in mice and is potentially to be used as a candidate vaccine for both prevention and therapy of HCV infection.