Objectives A potent HIV vaccine should overcome some limitations such as polymorphism of human HLA, the diversity of HIV-1 virus, and the lack of an effective delivery system. In this study, a DNA construct encoding Nef(60-84), Nef(126-144), Vpr(34-47), Vpr(60-75), Gp160(30-53), Gp160(308-323,) and P24(8-151) epitopes was designed using bioinformatics tools. The pcDNA3.1-nef-vpr-gp160-p24 and pcDNA3.1-nef constructs were prepared in large scale as endotoxin-free form. Moreover, the recombinant Nef-Vpr-Gp160-p24 polypeptide and Nef protein were generated inE. coli. These constructs were delivered using cell penetrating peptides (CPPs) in vivo, and immune responses were assessed for different modalities in BALB/c mice. Results The recombinant DNA constructs were confirmed as the similar to 867 bp and similar to 648 bp bands related tonef-vpr-gp160-p24 andnef genes on agarose gel. Moreover, the purified Nef-Vpr-Gp160-p24 polypeptide and Nef protein showed the similar to 32 kDa and similar to 30 kDa bands on SDS-PAGE, respectively. The results of immune responses indicated that the heterologous prime/boost regimens using both Nef-Vpr-Gp160-P24 and Nef antigens induced significantly the secretion of IgG2a, IgG2b, IFN-gamma and Granzyme B compared to other groups. The levels of Granzyme B in mice immunized with Nef antigen were higher than those immunized with Nef-Vpr-Gp160-P24 antigen. The CPPs showed the same potency with Montanide adjuvant for eliciting immune responses. Conclusions The heterologous prime/boost regimens for both antigens could significantly direct immune responses toward Th1 and CTL activity compared to other regimens. Comparing the efficiency of Nef-Vpr-Gp160-P24 and Nef constructs, the Nef-Vpr-Gp160-P24 constructs delivered by CPPs showed promising results as an HIV vaccine candidate.