Enhancement of cellular immune response to DNA vaccine encoding hepatitis C virus core and envelope 2 fusion antigen by murine Fms-like tyrosine kinase 3 ligand / 生物工程学报

ABSTRACT

Hepatitis C virus (HCV) is an important human pathogen that causes chronic liver disease worldwide. It is desirable to develop vaccines to prevent HCV infection, or at least to prevent progression to chronicity. We once constructed an optimized hepatitis C virus core and envelope 2 fusion antigen DNA vaccine, which could induce humoral and cellular immune responses against HCV core and E2 protein in BALB/c mice efficiently. Flt3 (Fms-like tyrosine kinase 3) -ligand has been identified as an important cytokine for the generation of professional antigen-presenting cells, particularly dendritic cells. We reasoned that a DNA vaccine coexpressing the antigen and FL may activate immune responses more effectually. In this study, The influence of FL on this HCV DNA vaccine was evaluated. The cDNA encoding signal peptide and extracellular domain of murine FL was inserted into the plasmid pST-CE2t, and the resulting plasmid pST-CE2t/FL was transfected into COS7 cells. The HCV core and E2 protein were detected by Western blotting, and the soluble murine FL was detected by ELISA. Eight-week-old female BALB/c mice were inoculated intramuscularly with 100 microg pST-CE2t, pST-CE2t/FL or mock vector, respectively, and boosted at the same dosage 3 weeks later. Anti-HCV core and E2 total IgG and isotypes were measured at weeks 1,3,5,7. Splenocyte proliferative response to recombinant HCV core and E2 protrein were detected at week 7. SP2/0 cells expressing HCV core protein were used as target cells for the detection of cytotoxic T lymphocyte (CTL) response. Western blot analysis showed that a protein band with molecular weight about 70 kD from lysate of COS7 cells transfected with plasmid pST-CE2t/FL could be detected by anti-HCV core or E2 monoclonal antibodies, which indicated that pST-CE2t could express glucosylated HCV core and E2 fusion protein. Murine FL could be detected in the culture supernatant of COS7 cells transfected with pST-CE2t/FL. Plasmid pST-CE2t immunized mice developed higher anti-HCV core and E2 IgG seroconversion rates and titers than pST-CE2t/FL group did at different various times, but the IgG2a/IgG1 ratio of anti-HCV E2 protein in pST-CE2t/FL group is much higher than pST-CE2t group. Splenocytes from pST-CE2t or pST-CE2t/FL immunized mice could proliferate with stimulation of HCV core or E2 protein in vitro, although pST-CE2t/FL group showed much stronger response. Splenocytes from mice immunized with pST-CE2t/FL induced 79.03% +/- 9.95% of target cell lysis at the effector/target ratio of 1001, which was significantly greater than the lysis (62.2% +/- 8.62%) observed in mice immunized with pST-CE2t. Our data demonstrated that the incorporation of FL can preferentially enhance the cellular response to this HCV fusion antigen DNA vaccine. In contrast, HCV specific antibodies were inhibited by FL in vaccinated mice. More and more data supports that recovery from acute HCV infection may depend upon the generation of broad-based cellular immune responses to viral proteins. So, FL may be of potential value as an adjuvant in the development of DNA-based immunization for prophylactic and therapeutic vaccine against HCV infection.