Agreement between an in-house replication competent and a reference replication defective recombinant virus assay for measuring phenotypic resistance to HIV-1 protease, reverse transcriptase, and integrase inhibitors

Abstract

Background: Although clinical management of drug resistance is routinely based on genotypic methods, phenotypic assays remain necessary for the characterization of novel HIV-1 inhibitors, particularly against common drug-resistant variants. We describe the development and assessment of the performance of a recombinant virus assay for measuring HIV-1 susceptibility to protease (PR), reverse transcriptase (RT), and integrase (IN) inhibitors.

Methods: The system is based on the creation of replication-competent chimeric viruses through homologous recombination between patient or laboratory virus-derived PCR fragments and the corresponding NL4-3 vector where the whole Gag-PR, RT-RNaseH or IN coding regions has been deleted through inverse PCR. The susceptibility to nucleoside (NRTIs) and non-nucleoside (NNRTIs) RT inhibitors and to IN inhibitors (INIs) is calculated through a single-round infection assay in TZM-bl cells, while protease inhibitor (PI) activity is determined through a first round of infection in MT-2 cells followed by infection of TZM-bl cells with MT-2 supernatants.

Results: The assay showed excellent reproducibility and accuracy when testing PI, NRTI, NNRTI, and INI susceptibility of drug-resistant clones previously characterized through the reference pseudoparticle-based Phenosense assay. The coefficient of interassay variation in fold change (FC) resistance was 12.0%-24.3% when assaying seven drug/clones pairs in three runs. FC values calculated by the Phenosense and in-house for 20 drug/clones pairs were in good agreement, with mean±SD ratio of 1.14±0.33 and no cases differing by more than twofold.

Conclusions: The described phenotypic assay can be adopted to evaluate the antiviral activity of licensed and investigational HIV-1 drugs targeting any of the three HIV-1 enzymes.

Keywords: HIV-1; HIV-1 inhibitors; drug resistance; phenotypic assay.

Figure 1

Overview of the in‐house recombinant virus phenotypic drug resistance assay. (A) Schematic representation of the deleted regions in the different plasmids used for generation of the recombinant virus. (B) Production of viral stocks upon transfection of 293FT cells, starting from infectious molecular clones or paired vector and insert DNA. (C) Schematic workflow of the MonoCycle and BiCycle assays for IC ₅₀ determination

Figure 2

Dose‐response curves for calculating the inter‐assay reproducibility of drug susceptibility testing, expressed as fold change (FC) with respect to the wild‐type reference virus. Numbers indicate the respective molecular clone as indicated in Table 1. R1, R2, and R3 indicate the replicate experiment done. ABC, abacavir; ATV, atazanavir, DRV, darunavir; EFV, efavirenz; RAL, raltegravir; RPV, rilpivirine; TDF, tenofovir disoproxil fumarate

Figure 3

Assessment of the accuracy of Mono/BiCycle assays compared to Phenosense assay. (A) Correlation between log‐transformed FC values obtained with the Mono/BiCycle assays and the reference Phenosense assay on a panel of drug‐resistance viruses. (B) Bland‐Altman plot showing the relationship between the average FC for the Phenosense and Mono/BiCycle assays and the ratio of the FC values obtained by the two systems