In an effort to achieve efficient and long-term inhibition of HIV-1 replication and propagation, a recombinant plasmid DNA was constructed, which can transcribe an almost full-length antisense HIV-1 RNA. HIV-1 pNL4-3 DNA was used for the construction of the recombinant antisense plasmid DNA. Co-transfection of recombinant antisense DNA and wild-type DNAs in 293 cells resulted in about 90% inhibition of reverse transcriptase (RT) activity produced from cloned DNAs of T-cell and macrophage-tropic viruses. Reduced RT activity was also seen upon transfecting the recombinant DNA into PM1 cells which had been infected with different T-cell tropic (LAI) and macrophage-tropic (BaL and JR-CSF) HIV-1 isolates. RT-PCR analysis indicated that the HIV-1 antisense RNAs could be co-packaged into virions along with the wild-type virus and that the antisense viral sequences could be transferred cell-free into naive cells and persist at least 30-days post-infection. The results indicate that the antisense HIV-1 plasmid DNA can inhibit various HIV-1 isolates representing the spectrum of HIV-1s commonly prevalent in North America. Current studies are directed towards investigation of different delivery systems for antisense RNAs including a compatible packaging system for HIV-1 antisense RNAs. Safety and Immunogenicity Studies of HIV-1 DNA Vaccines. Since both humoral and cellular and immune responses may be important against AIDS, we have constructed an HIV-1 plasmid DNA containing both the gag and env genes from pNL4-3 DNA under the regulation of different promoters. Analysis of immune responses in vaccinated monkeys indicated development of HIV-1 gag and env antibodies and cellular proliferative responses in rhesus and pig-tailed macaques. The presence of a strong promoter correlated with an earlier and higher antibody response than a weak promoter. Safety studies are underway to evaluate toxicity of DNA vaccination and presence of vaccine DNA sequences in the animals. Challenge studies will be initiated to evaluate the efficacy of the vaccines against HIV-1 in vaccinated pig-tailed macaques and recombinant SHIV in vaccinated rhesus macaques.