Open AccessAgreement 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
Author(s) -
Saladini Francesco,
Giannini Alessia,
Boccuto Adele,
Vicenti Ilaria,
Zazzi Maurizio
Publication year - 2018
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.22206
Subject(s) - reverse transcriptase , virology , integrase , recombinant dna , biology , virus , microbiology and biotechnology , integrase inhibitor , protease , viral replication , drug resistance , polymerase chain reaction , enzyme , human immunodeficiency virus (hiv) , viral load , genetics , gene , biochemistry , antiretroviral therapy
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 NL 4‐3 vector where the whole Gag‐ PR , RT ‐ RN aseH or IN coding regions has been deleted through inverse PCR . The susceptibility to nucleoside ( NRTI s) and non‐nucleoside ( NNRTI s) RT inhibitors and to IN inhibitors ( INI s) 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.