One limitation in the development of an improved cellular response needed for an effective HIV-vaccine is the inability to induce robust effector T-cells capable of suppressing a heterologous challenge. To improve cellular immune responses, we examined the ability of an optimized DNA vaccine to boost the cellular immune responses induced by a highly immunogenic Ad5 prime. Five Chinese rhesus macaques received pVax encoding consensus (con) gag/pol/env intramuscularly (IM) with electroporation followed by the Merck Ad5 gag/pol/nef vaccine. A second group of five animals were vaccinated with Merck Ad5 gag/pol/nef followed by pVax gag/pol/env. One year following vaccination, Ad5-prime DNA-boosted monkeys and four unvaccinated controls received an intrarectal challenge with 1000 ID50 SIV(mac)251. The quality and magnitude of the T-cell response was analyzed by ELISpot and polyfunctional flow cytometry. We observed that an Ad5-prime DNA-boost resulted in significantly elevated SIV-specific T-cell responses even compared with animals receiving a DNA-prime Ad5-boost. Ad5 prime DNA boosted animals were capable of suppressing a pathogenic SIV(mac)251 challenge. Peak control correlated with the expansion of HLA-DR(+) CD8(+) T-cells two weeks post-infection. These data illustrate that high optimization of a DNA vaccine can drive of immune responses primed by a robust vector system. This previously unachievable feature of these newly optimized DNAs warrants future studies of this strategy that may circumvent issues of serology associated with viral vector prime-boost systems.