Construction and characterization of an attenuated feline immunodeficiency virus proviral DNA vaccine that co-expresses interferon gamma.

摘要

Feline immunodeficiency virus (FIV) belongs to the lentivirus subfamily of retroviruses and is the etiological agent of a chronic, progressive immunodeficiency syndrome in domestic cats that is similar to AIDS in humans infected with human immunodeficiency virus (HIV). Therefore, FIV may serve as a valuable animal model for investigating HIV infection in humans and developing HIV vaccines and anti-viral therapies. Recent studies have demonstrated the feasibility of inducing protective immunity against homologous challenge in cats vaccinated with an attenuated FIV provirus encoding a deletion in the vif gene (FIVpPPR-Deltavif). It is hypothesized that, efficacy of an attenuated FIVDeltavif proviral DNA vaccine will be enhanced by co-expression of a Th1 cytokine such as interferon-gamma (IFN-gamma). As a first step to test this hypothesis, FIVDeltavif provirus that co-expresses feline IFN-gamma (FIVDeltavifATGgamma) was constructed and characterized. As a control for heterologous gene expression, a FIVDeltavif provirus encoding the green fluorescent protein (GFP) (FIVDeltavifATGgfp) was also constructed and characterized. Enhanced expression of the inserted genes was achieved by mutating two in-frame start codons at the 5' terminus of vif without altering the overlapping pol translation frame. In cell culture systems, both FIVDeltavif mutant proviruses expressed their respective heterologous genes and demonstrated severely restricted replication kinetics when compared to wild-type (WT) FIV-pPPR replication. To test FIVDeltavifATGgamma as a DNA vaccine, cats were inoculated by the intramuscular (IM) route with either FIV-pPPRDeltavif, FIVDeltavifATGgamma, or co-inoculated with FIV-pPPRDeltavif and a mammalian expression vector for IFN-gamma and evaluated for FIV-specific cellular and humoral immune responses. Immunization with FIVDeltavifATGgamma induced the greatest frequency of FIV-specific T-cell proliferation responses in vaccinated cats, whereas FIV-specific cytotoxic T-cell (CTL) responses were comparable between the different vaccine groups. Anti-Gag or anti-Env antibodies were not detected in any vaccinated cat. Virus was not detected by either virus isolation or viral DNA PCR assays in PBMC from cats from any experimental group post-inoculation with FIVDeltavif based DNA vaccine. All cats were challenged by the IM route at 13 weeks post-vaccination with ten 50% cat infectious doses (CID50) of FIV-PPR biological virus stock. T-cell proliferation responses and CTL responses measured after challenge were similar between vaccine groups. After challenge, anti-Gag antibodies and anti-Env antibodies were detected in vaccinated as well as control, unimmunized cats. However, a difference in the anti-Env antibody titers was not observed between vaccinated and unvaccinated control cats. Virus was isolated five weeks post-challenge from four out of five cats for each experimental vaccine group as well as the unvaccinated control group. In addition, plasma viral load, determined by quantitation of FIV RNA copy numbers, was not significantly different between vaccinated and unvaccinated control cats. These findings indicate that vaccination with FIV-pPPR-Deltavif did not protect cats against an early challenge with homologous virus at 13 weeks post-vaccination. Furthermore, co-expression of IFN-gamma did not enhance the efficacy of the FIVDeltavif DNA vaccine, although cellular immune responses measured at early time points post-challenge were greater in vaccinated cats. These results reveal that IFN-gamma is not an effective immunomodulator for the FIVDeltavif DNA vaccine tested in this study. Lastly, immune correlates of lack of protection observed with the different vaccine strategies were not clearly elucidated.