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New More Sensitive Monogram Tropism Assay;
Tweaked Trofile Assay Stronger in Spotting Dual/Mixed-Tropic Virus
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Targeting HIV Entry: 3rd International Workshop
December 7-9, 2007
Washington, DC
Mark Mascolini
A modified version of the Trofile HIV coreceptor tropism assay reclassified 8% of viral isolates from heavily pretreated candidates for the CCR5 antagonist maraviroc [1]. Nineteen of the 20 changes in determining HIV coreceptor preference reclassified CCR5-using HIV as dual/mixed (D/M)-tropic virus populations that can use either the CCR5 (R5) coreceptor or CXCR4 (X4) coreceptor. X4-using HIV does not respond to maraviroc of other CCR5 antagonists.
Monogram Biosciences' current Trofile assay can uncover small HIV caches that make up 10% of the viral population with 100% sensitivity--and caches that make up only 5% of the population with 85% sensitivity. Spotting more of these "minor variant" X4-using populations would be a plus because small pockets of X4 virus could expand during CCR5 antagonist therapy and overwhelm the regimen. In fact, people who apparently got into maraviroc trials with X4 virus did respond poorly.
Monogram investigators refined their Trofile test by manipulating viral input levels, cell coreceptor levels, and transfection and infection conditions. Then they tested a revised assay on viral populations collected from highly pretreated patients and on clones of R5, X4, and D/M virus.
The revamped assay heightened luciferase activity--the beacon Trofile reads to assign tropism--by a median 42-fold in nine D/M-using viral samples. In nine X4-tropic samples, the improved assay increased luciferase activity by a median 183-fold. Luciferase activity declined only 25-fold in five R5-using viral samples scrutinized by the revised assay.
Testing the tweaked Trofile assay in predefined mixtures of R5 and X4 clones, the Monogram team figured the revised test improved the sensitivity of spotting small X4 populations 3-fold to 10-fold. In a cluster of X4-using patient samples collected at different points, the enhanced assay permitted earlier detection of X4-using HIV compared with the standard assay. In one person with a small X4 population that the current assay sometimes spotted and sometimes did not, the rejiggered Trofile consistently called the samples D/M.
Focusing on 258 samples from 77 people, the revamped assay reclassified tropism in 8%, changing one X4 call and 19 R5 calls to D/M. These 20 changed calls involved 4 of the 77 people studied (5%).
Monogram will use the revised assay to check tropism calls in samples from completed clinical trials to determine whether changed tropism assignments could improve Trofile's clinical utility. The company is already applying to the US authorities who can sanction clinical use of the new assay.
Reference
1. Reeves J, Han D, Hunt P, et al. Enhancements to the Trofile HIV co-receptor tropism assay enable improved detection of CXCR4-using subpopulations and earlier detection of CXCR4-using viruses in sequential patient samples. HIV Entry: 3rd International Workshop. December 7-9, 2007. Washington, DC. Abstract 11.
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