A combined genotypic and phenotypic human immunodeficiency virus type 1 recombinant virus assay for the reverse transcriptase and integrase genes

The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG.

Section: Resultssupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

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Cross-Resistance Profile Determination of Two Second-Generation HIV-1 Integrase Inhibitors Using a Panel of Recombinant Viruses Derived from Raltegravir-Treated Clinical Isolates

Wesenbeeck¹,

Rondelez²,

Feyaerts³

et al. 2011

“…One amplicon per sample containing the reverse transcriptase-integrase (RT-IN) regions was sequenced and cloned into an HXB2 backbone. Recombinant viruses were titrated and subjected to an antiviral experiment with MT4-LTR-eGFP cells, as previously described (20). Briefly, MT4-LTR-eGFP cells were inoculated with a titrated amount of virus in the presence of 3-fold dilutions of the compound tested.…”

Section: Methodsmentioning

confidence: 99%

Broad Phenotypic Cross-Resistance to Elvitegravir in HIV-Infected Patients Failing on Raltegravir-Containing Regimens

Garrido¹,

Villacian²,

Zahonero³

et al. 2012

.5.01; Virco). A control group of patients failing on other regimens was similarly tested. Sixty-one samples were analyzed, 40 of which belonged to patients failing on RAL-containing regimens. Full RAL susceptibility was found in 20/21 controls, while susceptibility to EVG was diminished in 8 subjects, with a median fold change (FC) of 2. T he introduction of highly active antiretroviral therapy (HAART) has transformed the prognosis of HIV-positive patients, with dramatic improvements in survival. However, selection for drug resistance still represents a major challenge and promotes switches in therapeutic regimens for a substantial proportion of patients (3). Virologic failure with some antiretroviral drugs is associated with cross-resistance to agents within the same family, limiting rescue therapeutic options (16). Nevirapine and efavirenz are good examples of crossresistance between members of the same drug family, almost completely sharing resistance patterns. To a lesser extent, this also occurs with failures of some protease inhibitors (12) and nucleoside analogues.Integrase inhibitors (INIs) are a new and promising drug family for the treatment of HIV infection. Raltegravir (RAL) is the first-in-class approved drug for clinical use in both treatmentnaïve (11) and treatment-experienced (19) individuals. Elvitegravir (EVG) will most likely be the second INI compound to be marketed (4). Resistance to INIs has been shown to be driven by changes located mainly in the central domain of the viral integrase. The results of clinical trials and clinical experience have highlighted that failure on RAL-containing regimens is generally driven by the selection of the mutations Y143RHC, Q148HRK, and/or N155H, often accompanied by secondary changes (G140SA, E138K, and L74M, etc.) (2). However, a relatively high proportion of patients does not show such changes upon virologic failure on RAL-containing regimens. Although unidentified resistance mutations might exist, a recent report highlighted that the absence of INI resistance mutations in these individuals is explained mainly by poor drug compliance, given that RAL plasma levels were undetectable in most of them (6).Information about resistance to EVG in vivo is scarce. In the phase II GS-US-183-105 study (13), 28 individuals failed EVG after 24 weeks of treatment. Overall, 39% of them selected the E92Q mutation, 32% selected the Q148HRK mutation, and 25% selected the N155H mutation. Accordingly, broad cross-resistance between RAL and EVG should be expected. The aim of our study was to characterize RAL phenotypic susceptibilities in samples from a group of patients experiencing RAL failure, exploring whether mutations not previously involved in RAL resistance might be recognized and the extent of phenotypic cross-resistance to EVG. MATERIALS AND METHODSStudy population. HIV-1-infected individuals treated at several clinics in Spain who experienced virological failure on a RAL-containing antiretroviral regimen were identified during 2009. Virological failure was defined...

“…RT-IN amplicons were cloned into an HXB2D-based backbone with RT-IN deleted (56,58) using the In-Fusion Dry-Down PCR-cloning technology (Clontech-Westburg) following the manufacturer's protocol. The cloning mixtures were transformed into Max Efficiency Stbl2 cells (Invitrogen) using the manufacturer's procedure.…”

Section: Methodsmentioning

confidence: 99%

Secondary Integrase Resistance Mutations Found in HIV-1 Minority Quasispecies in Integrase Therapy-Naive Patients Have Little or No Effect on Susceptibility to Integrase Inhibitors

Ceccherini‐Silberstein

Baelen²,

Armenia

et al. 2010

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The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.