The main objective is to develop a form of HIV which will replicate only in the presence of the drug, doxycycline. Current live-viral vectors for HIV and other viruses cannot be conditionally controlled. These viruses, however attenuated, replicate in vivo to the extent permitted by the host's cells and immune system. Investigators have previously described a promoter-transactivator system that is active only in the presence of doxycycline. In this system the transactivator, RTTA, binds to the tet operator sequences only in the presence of doxycycline. When bound to the tet operator sequences, RTTA potently activates this promoter. HIV transcription is naturally under the control of the TAR-Tat interaction. Tat binds to TAR and activates the HIV promoter via interaction with host cell proteins. This form of activation is not amenable to exogenous control. HIV-Dox is being developed to allow conditional control of the activation process. In HIV-Dox, Tat cannot bind to TAR, which has been mutated. Without the TAR-Tat interaction, natural transcription is greatly reduced. Additional mutations in the U3 region of the LTR have been made to reduce the basal level of transcription even further. Upstream of the HIV TATAA box in HIV-Dox, tet operator sequences have been cloned. The gene for RTTA has been placed into the nef reading frame. In transient transfections, HIV-Dox virus production is dramatically upregulated in the presence of doxycycline (>100-fold increase). However, we have been unable to detect replication of HIV-Dox in CD4+ cells (by p24 ELISA or RT assay). The goals of the project are to determine why HIV-Dox does not replicate productively in T-cell lines and to repair this defect in order to obtain a doxycycline-inducible HIV vector. HIV-Dox would then be converted to SIV-Dox, which could be further developed in the macaque model of AIDS. After the safety and efficacy of SIV-Dox has been determined in macaques, HIV-Dox may be similarly developed as a potential vaccine in humans. In this scenario, the host, after receiving HIV-Dox, would be administered doxycycline for a defined period of time. While the host received doxycycline, HIV-Dox would replicate productively; this replication would induce an immune response. After the immune response has matured, the doxycycline administration would be discontinued and HIV-Dox would stop replicating. In this way, vaccine related disease could be reduced.
| Smith, S M; Khoroshev, M; Marx, P A et al. (2001) Constitutively dead, conditionally live HIV-1 genomes. Ex vivo implications for a live virus vaccine. J Biol Chem 276:32184-90 |
