Sensitivity of Phenotypic Susceptibility Analyses for Nonthymidine Nucleoside Analogues Conferred by K65R or M184V in Mixtures with Wild-Type HIV-1

Mark R. Underwood () 0 Lisa L. Ross 0 David M. Irlbeck 0 Peter Gerondelis 0 Elizabeth Rouse 0 Marty H. St. Clair 0 Lan Trinh 0 Neil Parkin 0 E. Randall Lanier 0 1 GlaxoSmithKline 0 Inc. 0 Research Triangle Park 0 North Carolina 0 Monogram Biosciences 0 South San Francisco 0 California 0 0 Received 17 March 2008; accepted 31 July 2008; electronically published 25 November 2008. Potential conflicts of interest: M.R.U. , L.L.R., D.M.I., P.G., E.R., M.H.S.C. , and E.R.L. are or have been employees of GlaxoSmithKline, and L.T. and N.P. are or have been employees of Monogram Biosciences. Presented in part: XIII International Drug Resistance Workshop , Tenerife Sur, Canary Islands, Spain, 8 -12 June 2004 (abstract 130). Financial support: GlaxoSmithKline and Monogram Biosciences 1 Present affiliation: Chimerix, Inc., Durham, North Carolina (E.R.L.). Disease Center of Excellence for Drug Discovery , 5 Moore Drive, RTP, NC 27709 -3398 Thymidine-sparing triple-nucleoside regimens have exhibited poor virologic response despite apparent phenotypic susceptibility to 2 of 3 regimen components at early time points. Phenotypic resistance masking by wild-type virus may explain this discrepancy. Consistent with this notion were (1) the presence of lowlevel nucleoside reverse-transcriptase inhibitor-resistant human immunodeficiency virus in subjects receiving failing first-line regimens consisting of tenofovir (TDF), abacavir (ABC), and lamivudine (3TC); (2) lower fold resistance associated with mixtures versus mutants in a clinical-isolate database; and (3) dosedependent changes in susceptibility to ABC, 3TC, TDF, and didanosine on titration of K65R and/or M184V with wild-type virus. These findings underscore the limitations of stand-alone phenotypic susceptibility measures and emphasize the importance of complementary and/or more sensitive techniques. (ClinicalTrials.gov identifier: NCT00053638.) Several clinical studies of thymidine-sparing (zidovudine or stavudine) triple-nucleoside-analogue therapy in treatment- - naive HIV-1infected subjects have been prematurely halted when an unacceptably high proportion of subjects (33% 63%) experienced early virologic failure. The regimens included the combination of tenofovir (TDF for studies in vivo, and TFV for in vitro assays), lamivudine (3TC), and didanosine (ddI) or abacavir (ABC) [15]. In these studies, high rates of selection for the reverse-transcriptase (RT) mutations K65R and M184I/V were observed by population sequencing. Phenotypic analyses indicated that, although the majority of samples had reduced susceptibility to 3TC, very few had apparent reduced susceptibility to other study drugs. To better understand why virologic failure occurred despite apparent susceptibility to 2 of the 3 drugs in the regimen, we evaluated minor variants via longitudinal clonal analysis. For this assessment, HIV genotypes and phenotypic susceptibilities were determined for 2 subjects with early virologic nonresponse who were receiving a TDF/ABC/3TC regimen in the ESS30009 study. To further understand the phenotypes of mutant plus wild-type mixtures, we determined the susceptibility of clinicalisolate samples with mixtures at positions 65 and 184 in RT across a large clinical database. Finally, we assessed the ability of a phenotypic assay to detect reduced nucleoside RT inhibitor (NRTI) susceptibility in samples containing defined mixtures of wild-type and K65R and/or M184V. Methods. The ESS30009 study was approved by the institutional review boards of each site, and all participants provided written informed consent. A limited number of the 41 subjects in the ESS30009 study receiving the TDF/ABC/3TC regimen who experienced virologic nonresponse had baseline and week 12 HIV genotypic and phenotypic data (PhenoSense GT; Monogram Biosciences) as well as plasma stored from weeks 2, 4, and 8. Additional genotypic analysis was done with week 2, 4, and 8 samples for 2 subjects by means of the TruGene HIV-1 genotyping assay (Bayer HealthCare). For clonal genotypic analysis, individual RT sequences from patient isolates were cloned from the TruGene RT PCR product by the ZERO Blunt Topo PCR cloning method (Invitrogen). The RT coding region from each individual bacterial colony was amplified by PCR and sequenced by standard methods. Phenotypic susceptibility analysis of NL4-3 based and HXB2-based plasmid mixtures with K65R and/or M184V mutations was performed at Monogram Biosciences, using the cellbased portion of the PhenoSense assay. Clinical cutoffs for reduced drug susceptibility (fold change in IC50 vs. reference [FC]) using the phenotypic assay are as follows: for TDF, 1.4; for ddI, 1.3; for ABC, 4.5; and for 3TC, 3.5. The sensitivity of the PhenoSenseGT assay for the detection of minority species on the basis of genotype is dependent on the relative chromatogram peak heights of each particular mutation. Detection of low levels of viral variants is optimized by combining computerized a (...truncated)