title: Rapid In Vitro Evaluation of Antiretroviral Barrier to Resistance at Therapeutic Drug Levels.
abstract: Failure of combination antiretroviral (ARV) therapy in HIV-infected patients is often associated with the emergence of drug resistance-associated mutations (RAMs). To facilitate analysis of the barrier to resistance at therapeutically relevant ARV concentrations, we performed fixed-dose in vitro HIV-1 drug resistance selection assays using the immortalized MT-2 T-cell line and primary human CD4+T cells with a panel of FDA-approved ARVs, each at their respective cell culture equivalent clinical trough concentration (CCE Cmin). At high multiples of its CCE Cmin, emtricitabine (FTC) selected for the rapid emergence of M184I/V, a result consistent with resistance emergence in vivo. While the rate of viral breakthrough in the presence of rilpivirine or efavirenz was delayed relative to FTC, both inhibitors selected for virus with known clinically relevant RAMs. No viral breakthrough was observed for the protease inhibitor atazanavir even at subtherapeutic drug concentrations, which is consistent with its previously characterized high in vivo barrier to resistance. Depending on assay conditions, treatment with integrase inhibitors elvitegravir and raltegravir resulted in breakthrough of both resistant and wild-type virus. The RAMs observed in drug selections were not detected above a 2% threshold by deep sequencing in the in vitro virus inoculum, and only rarely in isolates from treatment-naive HIV+ patients. These new viral breakthrough assays facilitate the analysis of multiple experimental replicates and conditions in parallel and provide a rapid quantitative means to evaluate drug resistance emergence at therapeutically relevant drug concentrations, which should facilitate the identification of new ARVs with a high barrier to resistance.
authors.affiliation: 1 Gilead Sciences, Inc. , Foster City, California.
authors.forename: Andrew
authors.initials: A
authors.lastname: Mulato
authors.affiliation: 1 Gilead Sciences, Inc. , Foster City, California.
authors.forename: Derek
authors.initials: D
authors.lastname: Hansen
authors.affiliation: 2 Seq-IT , Kaiserslautern, Germany .
authors.forename: Alex
authors.initials: A
authors.lastname: Thielen
authors.affiliation: 1 Gilead Sciences, Inc. , Foster City, California.
authors.forename: Danielle
authors.initials: D
authors.lastname: Porter
authors.affiliation: 1 Gilead Sciences, Inc. , Foster City, California.
authors.forename: George
authors.initials: G
authors.lastname: Stepan
authors.affiliation: 1 Gilead Sciences, Inc. , Foster City, California.
authors.forename: Kirsten
authors.initials: K
authors.lastname: White
authors.affiliation: 2 Seq-IT , Kaiserslautern, Germany .
authors.forename: Martin
authors.initials: M
authors.lastname: Daeumer
authors.affiliation: 1 Gilead Sciences, Inc. , Foster City, California.
authors.forename: Tomas
authors.initials: T
authors.lastname: Cihlar
authors.affiliation: 1 Gilead Sciences, Inc. , Foster City, California.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Yant
mesh_terms: D044966:Anti-Retroviral Agents / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002478:Cells, Cultured; D024882:Drug Resistance, Viral; D015497:HIV-1 / Q000187:drug effects*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D012641:Selection, Genetic
chemical_list: D044966:Anti-Retroviral Agents
keywords: HIV; antiretroviral therapy; drug discovery