Early detection of mixed mutations selected by antiretroviral agents in HIV-infected primary human lymphocytes

A growing concern in the pursuit of new therapies for HIV-1 infection is the potential for the virus to develop drug resistance. With the advent of modern antiretroviral therapy and the common use of combined modalities, it is difficult to identify in the clinic the mutations associated with a specific drug. In general, drug selection of mutants using a relevant cell system, such as primary human lymphocytes, is a good prognosticator of what will happen in humans. In this study, HIV-infected human peripheral blood mononuclear cells were exposed, at a concentration of 1- to 10-fold the median effective antiviral concentration, to the nucleosides (-)-beta-2',3'-dideoxy-3'-thia-5-fluorocytidine [(-)-FTC] (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC), 3'-azido-2',3'-dideoxyuridine (CS-87, AZDU), 3'-azido-2',3'-dideoxy-5-methylcytidine (CS-92, AZMC), 2',3'-didehydro-3'-deoxythymidine (d4T), beta-L-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (beta-L-D4FC), beta-L-2',3'-dideoxyadenine SATE[beta-L-ddAMP-bis(tbutylSATE)], beta-L-5-fluoro-2',3'-dideoxycytidine (L-FddC), and the protease inhibitors nelfinavir and amprenavir (VX-478). Virus from the culture supernatant was amplified by PCR and analysed by both HIV-1 reverse transcriptase and protease line probe assay. All the L-nucleoside analogues tested selected for the V184 mutation, including the L-pyrimidine nucleosides 3TC (-)-FTC, beta- L-FddC, beta-L-D4FC and the beta-L-purine nucleoside. beta-L-D4FC also selected for K/R65 in addition to V184, indicating that these two mutations are linked and compatible in vitro. No pattern of mutations leading to resistance or reduced susceptibility was discerned with d4T. Rapid genotyping analysis revealed the different kinetics and mutations obtained by in vitro selection in HIV-infected cells exposed to nucleoside analogues and protease inhibitors.