Conditional-live HIV-1 variants. (A) In the HIV-rtTA virus, the Tat-TAR axis of transcription regulation has been inactivated by mutation of both Tat and TAR (crossed boxes), and transcription and replication of the virus was made DOX dependent by introducing tetO elements in the long terminal repeat (LTR) promoter region and replacing the Nef gene with the rtTA gene. A T20-dependent HIV-rtTA-T20 variant was constructed by introduction of the Val-554-Ala and Asn-642-Lys mutations in the Env-gp41 domain. This construction caused an additional Asn-641-Ser mutation that does not affect virus replication (5) (unpublished results). (B and C) Replication of HIV-rtTA and HIV-rtTA-T20. SupT1 T cells were transfected with 1 μg of the molecular clones and cultured in the presence (+) or absence (-) of 100 ng of DOX/ml and 500 ng of T20/ml (as described previously [5, 12]). Virus replication was monitored by CA-p24 enzyme-linked immunosorbent assay (ELISA) on culture supernatant samples. A typical experiment is shown. Similar results were obtained in independent experiments that were started by either infection or transfection of SupT1 cells.

Vaccination strategy with the DOX- and T20-dependent HIV-1 virus. (A) Upon prime vaccination with HIV-rtTA-T20, replication can be temporarily activated and controlled to the extent that is needed for induction of the immune system by transient DOX and T20 administration. After priming of the immune system, DOX and T20 are withdrawn to stop virus replication. If this priming is not sufficient to confer protective immunity, or if it is necessary to refresh immunological memory, a booster vaccination can be achieved by the administration of DOX, which will activate virus production in cells carrying the integrated HIV-rtTA-T20 provirus, yet the infection of new cells and virus spread will be blocked in the absence of T20. (B) To simulate this vaccination scenario, SupT1 cells were transfected with HIV-rtTA-T20 and cultured in the presence of both DOX and T20 (different symbols represent different cultures). At day 3, the culture medium was replaced with medium without DOX and T20 (-/-). At day 35, each culture was split in four samples, to which either DOX (100 ng/ml), T20 (500 ng/ml), DOX plus T20, or no inducer (-/-) was added. Virus production was monitored by CA-p24 ELISA on culture supernatant samples.