The SV40t polyadenylation and splicing signals of the pAEC plasmid vectors were replaced by synthetic intron and synthetic rabbit beta globin-based termination/polyadenylation sequences, and 5, 10, and 20 copies of the 5'-AACGTT-3' CpG motif were inserted. Balb/c mice were immunized by intramuscular injection of 200 microg of each plasmid, coding for the HIV-1 multiepitope TAB9, under the control of the human cytomegalovirus promoter. After three doses of DNA, a fourth boost with plasmid DNA or a TAB9-expressing recombinant fowlpox virus rFPTAB9LZ was administered. ELISA and ELISPOT assays were conducted for antibody and IFN-gamma-secreting cell-mediated responses' evaluation against the whole TAB9 and the TAB9's IIIB V3 peptide, respectively. Serum IgG antibodies were not detected. Effector IFN-gamma-secreting responses were only detected on the animals receiving the new set of DNA constructs, alone or in combination with a recombinant virus boost, with or without in vitro re-stimulation. The response was dependent on the new transcriptional unit and influenced by the number of CpG motifs. We showed that plasmid backbone optimization based on these two factors could enhance the response against a multiepitope-based DNA vaccine. A new family of plasmid vectors is also available for evaluation with desired antigens.