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48th Annual ICAAC / IDSA 46th Annual Meeting
October 25-28, 2008
Washington, DC
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Validation of an Enhanced Sensitivity Trofile™ HIV-1 Co-receptor Tropism Assay
  Reported by Jules Levin
ICAAC/IDSA Oct 2008 Wash DC
Lan Trinh, Dong Han, Wei Huang, Terri Wrin, Jeffrey Larson, Linda Kiss, Eoin Coakley, Christos Petropoulos, Neil Parkin, Jeannette Whitcomb, and Jacqueline Reeves* Monogram Biosciences, South San Francisco, CA, USA
Enhanced Trofile has improved sensitivity to detect low levels of CXCR4-using variants in env clone mixtures and patient env populations compared to the original assay, while assay accuracy, precision and reproducibility are maintained.
Enhanced Trofile has increased utility for selecting patients for CCR5 antagonist therapy and replaced the original Trofile assay for patient selection in June 2008.
Several HIV entry inhibitors which block infection via CCR5 are in clinical development and maraviroc, a CCR5 antagonist, has been approved for use in treatment-experienced patients with CCR5-tropic (R5) virus.
TrofileTM is a clinically validated HIV co-receptor tropism assay (Monogram Biosciences) (Figure 1) for selecting patients for appropriate treatment with CCR5 antagonists. Trofile determines whether a patient's viral population is CCR5 (R5), CXCR4 (X4) or dual (R5/X4)/mixed (D/M)-tropic and has demonstrated utility in clinical trials of CCR5 antagonists including maraviroc and vicriviroc.
In mixed envelope (env) populations, the original Trofile assay was validated to detect minor R5 and X4 variants at 10 and 5% of the population with 100 and 85% sensitivity, respectively (Figure 2) (levels below 5% were not tested) (1).
Low level CXCR4-using variants below the detection limit of the original Trofile assay can sometimes be identified by clonal analysis of patient Env populations and may be selected following therapy with CCR5 antagonists.
As the detection of CXCR4-using virus below the original Trofile assay sensitivity limit may further optimize patient selection (2,3), we validated an enhanced sensitivity version of Trofile that allows an average ~30-fold improved detection of X4 variants in env clone mixtures (100% sensitivity at detecting 0.3% X4 Envs) and earlier detection of minor CXCR4-using subpopulations in longitudinal samples from PR/RT inhibitor experienced patients (4,5,6,7).
Experiments were performed to validate the performance of enhanced Trofile for patient management applications in compliance with CAP and CLIA regulations. The co-receptor tropism of a panel of well characterized viral isolates and patient-derived HIV-1 envelopes (Envs) were evaluated to assess assay accuracy. Precision and reproducibility were assessed by replicate testing. Minor variant sensitivity was determined using mixtures of paired R5 and X4 env clones derived from 4 patients. R5 and X4 pairs were selected to exhibit similar infectivity of CCR5 and CXCR4 expressing cells, respectively.
See figures below

Figure 2. Enhanced Trofile accurately determined the coreceptor tropism of a panel of 46 well characterized Envs representing multiple subtypes and clonally analyzed patient Env populations. Intra-assay precision (100%) and interassay reproducibility (99%) were demonstrated from concordant results for 135/135 and 228/230 pair-wise comparisons of R5, X4 and DM Envs clones and from repeat testing of 46 patient Env populations, respectively. Assay validation performance characteristics were equivalent between the original and enhanced Trofile assays, with the exception that enhanced Trofile is validated to detect 0.3% X4 Envs with 100% sensitivity, compared to 10% minor variant with 100% sensitivity for original Trofile.
Figure 3. Across 288 assays to evaluate minor variant sensitivity, enhanced Trofile detected X4 clones in 100% of assays when present at 0.3% and in 81% of assays at 0.1%. R5 clone detection was also improved compared to original Trofile, with 5% R5 clones detected in 100% of assays and 1% in 94% of assays.
Figure 4. The lower limit of X4 and R5 variant detection was env clone pair (patient) dependent and ranged from 100% sensitivity to detect 0.003-0.3% X4 clones and 0.3-5% R5 clones by enhanced Trofile.
Figure 5. Enhanced Trofile detected CXCR4-use in 14/18 clonally analyzed patient samples with CXCR4-using variants below the level of detection of the original Trofile assay and detected CCR5-use in 1/4 clonally analyzed X4 patient samples.




Enhanced Trofile detected CXCR4-use in 14/18 clonally analyzed patient viruses with CXCR4-using Envs below the detection limit of original Trofile and detected CCR5-use in 1/4 patient samples with X4 viruses by original Trofile and from analysis of approximately 48-96 clones
(1). Whitcomb et al. AAC Vol. 51, p. 566-575, 2007.
(2). Reeves et al. 15th CROI, Abstract 869, 2008.
(3). Su et al. Antiviral Therapy Vol. 13, Suppl 3, p. A98, 2008.
(4). Reeves et al. 47th ICAAC, H-1026, 2007.
(5). Reeves et al. 11th European AIDS Conference, P1.2/03, 2007.
(6). Reeves et al. 3rd International Workshop on Targeting HIV Entry, 11, 2007.
(7). Trinh et al. Antiviral Therapy Vol. 13, Suppl 3, p. A128, 2008.