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Updated: May 13, 2026
Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach
Published on: December 1, 2011
Sensitive cell-based assay for determination of human immunodeficiency virus type 1 coreceptor tropism
Jan Weber1, Ana C Vazquez, Dane Winner
1Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic.
Abstract:
CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.
Insights
A new assay, VERITROP, accurately detects HIV-1 coreceptor tropism, identifying non-R5 variants crucial for guiding CCR5 antagonist therapy in HIV patients.
Area of Science:
- Virology
- Immunology
- Antimicrobial Resistance
Background:
- CCR5 antagonists are a key antiretroviral drug class for HIV treatment.
- Accurate determination of HIV-1 coreceptor tropism (R5 vs. non-R5) is essential for selecting appropriate CCR5 antagonist therapy.
- Existing tropism assays have limitations in detecting minority non-R5 variants.
Purpose of the Study:
- To develop and validate a novel phenotypic assay, VERITROP, for sensitive and accurate determination of HIV-1 coreceptor tropism.
- To evaluate VERITROP's performance against established tropism assays and sequencing methods.
- To assess VERITROP's utility in predicting treatment outcomes with CCR5 antagonist drugs.
Main Methods:
- Development of a proprietary yeast-based vector system for HIV-1 env gene cloning.
- Utilized a sensitive cell-to-cell fusion assay to detect R5 and non-R5 viral variants.
- Compared VERITROP results with the original Trofile assay, population sequencing, and 454 pyrosequencing using various bioinformatics tools.
Main Results:
- VERITROP demonstrated high analytical sensitivity (0.3%) for detecting minority non-R5 variants across different viral loads and subtypes.
- Pilot study showed 73.7% concordance with the original Trofile assay, with VERITROP identifying 19 additional non-R5 cases.
- VERITROP showed better concordance with deep sequencing/Geno2Pheno and accurately predicted maraviroc treatment success, similar to 454 sequencing.
Conclusions:
- VERITROP is a sensitive and prevalidated phenotypic assay for HIV-1 coreceptor tropism determination.
- VERITROP effectively detects clinically relevant minority non-R5 variants missed by other methods.
- VERITROP has the potential to guide the use of novel HIV coreceptor antagonists and improve patient management.
