Virol 79:3419-3428, 2005). To make this DNA safer, we deleted two more genes, the integrase gene and vif, along with the 3 long terminal repeat. We also replaced the gag, pro, and nef genes (SIVmac239 origin) with those of human immunodeficiency virus (HIV) type 1 strain SF2. The resultant construct, designated ⌬4SHIV KU2 DNA, was used in this study to evaluate gene expression and immunogenicity in BALB/c mice. DNA-transfected human embryonic kidney epithelial cells (HEK 293) produced all of the major viral proteins and released p24 in the supernatant for 12 days. Inoculation of the vaccine DNA into the gastrocnemius muscles resulted in intense mononuclear cell infiltration at the inoculated sites and the production of viral p24 in myocytes, in infiltrating mononuclear cells, and in cells in the spleen and draining lymph nodes between 3 and 10 days postinoculation. Expression of p24 in the muscle cells peaked at day 7 and became undetectable after day 12. The same 12-day period of expression of p24 was observed in mice that were given a second injection 4 weeks after the first. Evaluation of immune responses in BALB/c mice revealed that the DNA induced enzyme-linked immunospot and antigen-specific proliferative cell-mediated immunity responses. The responses were stronger in mice that were coinjected with a second plasmid expressing granulocyte-macrophage colony-stimulating factor. Since new waves of viral antigen production could be induced with each boosting injection of the vaccine DNA, this DNA could be a safe and efficient agent to induce long-term protection against HIV.An effective and safe vaccine against human immunodeficiency virus (HIV)/AIDS still remains elusive even after more than a decade of intense research. Although live vaccines are highly effective in preventing AIDS in macaques, they are not acceptable for use in humans because of concerns about reversion to pathogenicity by mutation or recombination. An alternative strategy using recombinant viral envelope glycoprotein was not effective in clinical trials. Further alternative strategies using viral DNA as a vaccine were therefore adopted. Initial clinical trials of certain of the DNA vaccines in humans have demonstrated only a limited degree of immunogenicity thus far (for a review, see reference 20), necessitating further improvement in DNA vaccine construction strategies. Attempts to improve the efficiency of DNA vaccines have been made either by optimization of HIV genes for codon usage in mammalian cells (4,7,18,44,45) or by modifying RNA structures through nucleotide changes facilitating high antigen expression in a Rev-independent manner (32). Other strategies tried to potentiate DNA vaccines by modulating and enhancing host immune response with the help of cytokine/chemokine adjuvants and T-cell costimulatory molecules (9,10,14,32) or by different enhancers/promoters such as cytomegalovirus (37) or ␤-actin or muscle-specific desmin promoter (13) to obtain high expression of one or two fused genes of HIV (26). The use of these DNA moie...