title: Sensitive cell-based assay for determination of human immunodeficiency virus type 1 coreceptor tropism.
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.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Weber
authors.forename: Ana C
authors.initials: AC
authors.lastname: Vazquez
authors.forename: Dane
authors.initials: D
authors.lastname: Winner
authors.forename: Richard M
authors.initials: RM
authors.lastname: Gibson
authors.forename: Ariel M
authors.initials: AM
authors.lastname: Rhea
authors.forename: Justine D
authors.initials: JD
authors.lastname: Rose
authors.forename: Doug
authors.initials: D
authors.lastname: Wylie
authors.forename: Kenneth
authors.initials: K
authors.lastname: Henry
authors.forename: Alison
authors.initials: A
authors.lastname: Wright
authors.forename: Kevin
authors.initials: K
authors.lastname: King
authors.forename: John
authors.initials: J
authors.lastname: Archer
authors.forename: Eva
authors.initials: E
authors.lastname: Poveda
authors.forename: Vicente
authors.initials: V
authors.lastname: Soriano
authors.forename: David L
authors.initials: DL
authors.lastname: Robertson
authors.forename: Paul D
authors.initials: PD
authors.lastname: Olivo
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Arts
authors.forename: Miguel E
authors.initials: ME
authors.lastname: Quiñones-Mateu
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D002459:Cell Fusion; D002460:Cell Line; D003510:Cyclohexanes; D016679:Genome, Viral; D005838:Genotype; D057809:HEK293 Cells; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D000077592:Maraviroc; D010865:Pilot Projects; D012367:RNA, Viral / Q000235:genetics; D019713:Receptors, CCR5 / Q000235:genetics*; D019718:Receptors, CXCR4 / Q000235:genetics*; D015728:Receptors, HIV / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D014230:Triazoles; D056189:Viral Tropism; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics
chemical_list: D019380:Anti-HIV Agents; C527276:CXCR4 protein, human; D003510:Cyclohexanes; D012367:RNA, Viral; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D015728:Receptors, HIV; D029701:Saccharomyces cerevisiae Proteins; D014230:Triazoles; C554167:URA3 protein, S cerevisiae; D054299:env Gene Products, Human Immunodeficiency Virus; D000077592:Maraviroc