The aim of this application is to improve the efficacy of DNA vaccines to HIV-1 by optimizing vector driven transgene expression. We hypothesize that the viral promoters used in current DNA vaccines for regulation of antigen expression are down-regulated by cytokines such as interferon (IFN)-gamma which are rapidly induced in vivo due to the immunostimulatory activity of unmethylated CpG sequences present in the bacterial backbone of expression vectors. As such sequences are needed to initiate innate immune responses which in turn promote activation of antigen specific immunity we propose to test the suitability of alternative promoters which are upregulated by cytokines for use in DNA vaccines. One such promoter is the human IRF-1 promoter which was shown to exhibit enhanced activity in bone marrow derived cells upon addition of IFN-gamma, an inverted repeat domain (from -130 to -106) of the IRF-1 promoter positioned immediately upstream of a viral promoter also confers IFN-gamma inducibility to this viral promoter. In the 1st aim we will test the effect of cytokines, most notably IFN-gamma on antigen expression in cells, including dendritic cells, transiently transfected with vectors based on the CMV promoter (control), the IRF-1 promoter or the inverse repeat domain positioned upstream of the CMV promoter. In the second part of this aim we test the levels of transcript expression in muscle and lymph nodes of mice, including IFN-gamma knock-out (GKO) mice, inoculated with these constructs, using plasmids expressing gp160 of HIV-1 to confirm that in animals antigen production by the different constructs is regulated either negatively (CMV promoter) or positively (IRF-1 promoter) by IFN-gamma. In the 2nd aim we will test the 3 different constructs for induction of T and B cell responses in normal and in GKO mice. The effect of genetic adjuvants, that is vector encoding cytokines given concomitantly with the DNA vaccine will be investigated focusing on those that affect the IFN-gamma activation pathway. Assuming that even an optimized DNA vaccine might lack the potency to induce a sufficiently robust immune response to ward off a chronic infection with HIV-1 we will in our 3rd aim combine the optimized DNA vaccine with a traditional viral recombinant vaccine in a prime/boost regimen.
