title: Development of a phenotypic susceptibility assay for HIV-1 integrase inhibitors.
abstract: Phenotypic resistance analysis is an indispensable method for determination of HIV-1 resistance and cross-resistance to novel drug compounds. Since integrase inhibitors are essential components of recent antiretroviral combination therapies, phenotypic resistance data, in conjunction with the corresponding genotypes, are needed for improving rules-based and data-driven tools for resistance prediction, such as HIV-Grade and geno2pheno[integrase]. For generation of phenotypic resistance data to recent integrase inhibitors, a recombinant phenotypic integrase susceptibility assay was established. For validation purposes, the phenotypic resistance to raltegravir, elvitegravir and dolutegravir of nine subtype-B virus strains, isolated from integrase inhibitor-naïve and raltegravir-treated patients was determined. Genotypic resistance analysis identified four virus strains harbouring RAL resistance-associated mutations. Phenotypic resistance analysis was performed as follows. The HIV-1 integrase genes were cloned into a modified pNL4-3 vector and transfected into 293T cells for the generation of recombinant virus. The integrase-inhibitor susceptibility of the recombinant viruses was determined via an indicator cell line. While raltegravir resistance profiles presented a high cross-resistance to elvitegravir, dolutegravir maintained in-vitro activity in spite of the Y143R and N155H mutations, confirming the strong activity of dolutegravir against raltegravir-resistant viruses. Solely a Q148H+G140S variant presented reduced susceptibility to dolutegravir. In conclusion, our phenotypic susceptibility assay permits resistance analysis of the integrase gene of patient-derived viruses for integrase inhibitors by replication-competent recombinants. Thus, this assay can be used to analyze phenotypic drug resistance of integrase inhibitors in vitro. It provides the possibility to determine the impact of newly appearing mutational patterns to drug resistance of recent integrase inhibitors.
authors.affiliation: Institute of Virology, University of Cologne, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Heger
authors.affiliation: Institute of Virology, University of Cologne, Germany.
authors.forename: Alexandra Andrée
authors.initials: AA
authors.lastname: Theis
authors.affiliation: Institute of Virology, University of Cologne, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Remmel
authors.affiliation: Medical Center for Infectiology, Berlin, and Medical Laboratory Stendal, Stendal, Germany.
authors.forename: Hauke
authors.initials: H
authors.lastname: Walter
authors.affiliation: Department of Computational Biology and Applied Algorithmics, Max Planck Institute for Informatics, Saarbrücken, Germany.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Pironti
authors.affiliation: Institute of Virology, University of Cologne, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Knops
authors.affiliation: Institute of Virology, University of Cologne, Germany.
authors.forename: Veronica
authors.initials: V
authors.lastname: Di Cristanziano
authors.affiliation: Department of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, University Hospital Düsseldorf, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Jensen
authors.affiliation: Department of Dermatology and Venerology, University Hospital Duisburg-Essen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Esser
authors.affiliation: Institute of Virology, University of Cologne, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Kaiser
authors.affiliation: Institute of Virology, University of Cologne, Germany. Electronic address: nadine.luebke@med.uni-duesseldorf.de.
authors.forename: Nadine
authors.initials: N
authors.lastname: Lübke
mesh_terms: D024882:Drug Resistance, Viral / Q000235:genetics; D005838:Genotype; D015658:HIV Infections / Q000821:virology; D019427:HIV Integrase / Q000235:genetics* / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D010078:Oxazines; D010641:Phenotype; D010879:Piperazines; D011728:Pyridones; D015363:Quinolones / Q000494:pharmacology; D000068898:Raltegravir Potassium / Q000494:pharmacology; D000099226:Dolutegravir
chemical_list: D019427:HIV Integrase; D019428:HIV Integrase Inhibitors; D006575:Heterocyclic Compounds, 3-Ring; D010078:Oxazines; D010879:Piperazines; D011728:Pyridones; D015363:Quinolones; D000068898:Raltegravir Potassium; D000099226:Dolutegravir; C509700:elvitegravir; C514801:p31 integrase protein, Human immunodeficiency virus 1
keywords: Cross-resistance; Dolutegravir; Geno2pheno; HIV-1; Integrase; Phenotypic resistance