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187 Construction and Characterization of Attenuated Feline Immunodeficiency Virus Proviral DNA Vectors Expressing Immunomodulators
S. Gupta*, J. Byerly, and E. Sparger
Univ. of California, Davis, USA
Background: Recent studies have demonstrated the feasibility of inducing protective immune responses in cats by inoculation with attenuated proviral DNA vaccines. It is hypothesized that efficacy of an attenuated FIV proviral DNA vaccine will be enhanced by coexpression of immunomodulators. To assess this hypothesis, a novel infectious FIVDELTAvif proviral vector expressing IFNgamma was constructed and characterized in vitro. As a control for heterologous gene expression, a similar vector encoding GFP was also constructed. Furthermore, a mammalian expression vector expressing IFNgamma under the control of the FIV rev-response element (RRE), such that expression of IFNgamma is dependent on FIV Rev supplied in trans, was constructed and characterized.
Methods: (1) FIV pPPR-deltavif mutant proviruses encoding feline IFNgamma or GFP were constructed. The ATG start codon of vif and another ATG within vif, 5 of the inserted heterologous gene were mutated. These vectors were tested for IFNgamma or GFP expression in Cos cells by transfection and immunocytochemistry or transfection and flow cytometry, respectively. In addition, IFNgamma mRNA was assessed in transfected cells by TaqMan. (2) Replication of these proviral vectors was evaluated in feline PBMC by FIV-specific Gag antigen ELISA. (3) An expression vector encoding IFNgamma flanked by the tat/rev splice donor sequence of HIV-1 SF-2 on the 5 end, and the FIV-RRE, INS sequences (p15 of FIV), and the tat/rev splice acceptor site of HIV-1 SF-2 on the 3 end was constructed and characterized as above. (4) Antiviral activity of IFNgamma expressed from these viral vectors was evaluated by measuring inhibition of cytopathic effects of VSV on feline FC9 cells.
Results and Conclusions: FIV pPPR-deltavif proviruses encoding feline IFNgamma or GFP demonstrated severely restricted replication in vitro when compared with WT-FIV. These vectors stably expressed their respective heterologous genes as well as FIV Gag and Env. An expression vector encoding IFNgamma under the control of FIV-RRE expressed IFNgamma in vitro when cotransfected with WT-FIV, whereas IFNgamma expression was reduced in the absence of WT-FIV, demonstrating that expression of IFNgamma is dependent on FIV Rev. IFNgamma expressed by these viral vectors exhibited biological activity.
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