Rapid, phenotypic HIV-1 drug sensitivity assay for protease and reverse transcriptase inhibitors

Background: Development of drug resistance is one of the major reasons for the failure of antiretroviral therapy of HIV-1 infection. Knowing the drug sensitivity-resistance profile of viruses present in a patient prior to tre atment or change in treatment could help to optimize therapy. Objective: Development of a rapid standardized phenotypic HIV-1 drug sensit ivity assay for protease (PR) and reverse transcriptase (RT) inhibitors. Design: The PR gene (codons 1-99) and the 5' part of the RT gene (codons 1- 300) of HIV-1 is amplified from the plasma of infected individuals by RT-PC R and ligated into a proviral clone of HIV-1 containing a deletion of the P R gene and the 5' part of the RT gene. Bacteria are transformed with the li gation product and plasmid DNA is prepared from a library of transformed ba cteria. The plasmid DNA. is transfected into 293 T cells and recombinant vi rus is harvested from the supernatant of the transfected cells 2 days after transfection. The sensitivity of the recombinant virus is determined with the help of a sensitive indicator cell line. Results: Recombinant viruses were generated with high efficiency. Determina tion of the drug sensitivity of the recombinant viruses with an indicator c ell line was highly reproducible. The recombinant viruses accurately reflec ted the sensitivity-resistance profile of the parental viruses. The phenoty pic drug sensitivity determined by this assay correlated well with the trea tment history of patients. Conclusion: This assay system should allow rapid, high-throughput analyses of phenotypic HIV-I drug sensitivity for PR and RT inhibitors. Due to the e fficient generation of recombinant viruses, propagation of the recombinant viruses in cell culture is not required prior to the determination of the s ensitivity of the recombinant viruses. The risk of selecting fitter non-res istant viruses due to culture conditions is minimized. (C) 1999 Elsevier Sc ience B.V. All rights reserved.