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.

Keywords: Cross-resistance; Dolutegravir; Geno2pheno; HIV-1; Integrase; Phenotypic resistance.