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189 Constitutively Dead, Conditionally Live HIV-1 Genomes: Ex Vivo Implications for a Live-Virus Vaccine
M. Khoroshev*1, P. Marx2,3, J. Orenstein4, K.-T. Jeang5, and S. Smith1
1St. Michael’s Med. Ctr. and New Jersey Med. Sch.-UMDNJ, Newark, USA; 2Tulane Region. Primate Res. Ctr., Covington, LA, USA; 3The Rockefeller Univ., New York, NY, USA; 4George Washington Univ. Sch. Med., Washington, DC, USA; and 5NIAID/NIH, Bethesda, MD, USA
Background: In an attempt to create a safer form of HIV-1 vaccine, constitutively dead, conditionally live (CDCL) genomes were constructed. The genomes are constitutively defective for the Tat/TAR axis and are conditionally dependent on tetracycline/doxycycline for attenuated replication.
Methods: TAR function was inactivated by a 9-bp mutation. A Tet-operator fragment (7 inverted repeat sequences of the operator O2 of Tn10) was ligated upstream TATAA box of pNL4-3 clone, stripped of U3 elements. A cDNA for the reverse tetracycline-controlled trans-activator, RTTA, was substituted in place of nef-coding sequence. Genotypically, the genomes were tat(+)tar(-)nef(-)Sp1(-) [pHIVDoxT] or Sp1(+) [pHIVDoxSp]. Results: HIVDoxT and HIVDoxSp are fully characterized and are similar to NL4-3. (1) Electron microscopy shows normal maturation and release of virions from transfected 293T cells; (2) p24-ELISA and immunoblots of transfected 293T cell lysates confirm quantitatively normal expression of viral proteins; (3) reverse transcriptase, RT, is functional as determined by an enzymatic RT assay; and (4) viral RNA is packaged with the same efficiency as seen in NL4-3, based on quantitative RT-PCR. Doxycycline induces virion production over 25-fold in transfected 293T cells. After infection of Jurkat cells, HIVDoxT and HIVDoxSp genomes are detectable by PCR; however, Gag production is undetectable. Removing the RTTA sequence from the proviral genome allows more robust replication in Jurkat cells, which stably express either RTTA or TTA. Passaging of RTTA(() HIVDoxSp resulted in significant changes in U3, but TAR did not revert.
Conclusions: Robust expression of viral proteins of CDCL genomes coupled with their highly attenuated replication phenotype may describe an optimal combination for a strong and satisfactory HIV-1 vaccine. At present time the current design is under evaluation for the cell-mediated immunity through in vivo inoculation of CDCL genomes into nonhuman primates.
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