MultiCode-RTx Real-Time PCR System for Detection of Subpopulations of K65R Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant Viruses in Clinical Samples

Antimicrob Agents Chemother

et al. 2015

Self Cite

T he majority of marketed antiretroviral (ARV) inhibitors for treatment of HIV-1 infection target one of three enzymes essential for viral replication: protease (PR), reverse transcriptase (RT), or integrase (IN). Initial ARV therapy recommended by the current treatment guidelines consists of a combination of three inhibitors from at least two drug classes, typically, two nucleos(t)ide RT inhibitors (NRTIs) plus a ritonavir-boosted PR inhibitor (PI), nonnucleoside RT inhibitor (NNRTI), or IN strand transfer inhibitor (INSTI) (1-5). While ARV therapy successfully suppresses viral replication in most cases, the emergence of drug resistance mutations may occur and constitutes a major limitation to long-term treatment efficacy. Resistance-associated mutations (RAMs) often accumulate during virologic failure, potentially reducing viral susceptibility to multiple drugs along with reducing viral replicative fitness. However, additional accessory mutations that may compensate for this fitness defect, with or without further diminishing drug susceptibility, can also develop within the same coding region of the target enzyme (6-9).Single-tablet regimens (STRs) can offer complete ARV therapy in a once-daily pill. The first INSTI-based STR combines elvitegravir (EVG) with the pharmacokinetic enhancer cobicistat (COBI) and the NRTIs emtricitabine (FTC) and tenofovir (TFV) disoproxil fumarate (TDF) (EVG-COBI-FTC-TDF) (10). In the phase III studies of EVG-COBI-FTC-TDF, 18 of the 701 total subjects treated with EVG-COBI-FTC-TDF (2.6%) developed RAMs through 144 weeks (11)(12)(13)(14). The most common pattern of viral resistance contained the FTC primary resistance substitution M184V in RT (RT-M184V) coupled with the EVG primary resistance substitution E92Q in IN (IN-E92Q) (8 subjects) (13-15). The TFV resistance substitution K65R in RT (RT-K65R) also occurred in combination with the M184V and E92Q substitutions in two of these cases. Clonal sequence analysis of patient-derived viral isolates revealed that these substitutions often occurred together on the same viral genomes (15). While IN-E92Q, RT-M184V, and RT-K65R have been previously characterized individually and with other substitutions in the same coding region, the impact of these three substitutions combined in the same genome on drug susceptibility or viral fitness has not been studied.Since HIV-1 IN and RT enzymes are expressed together in a polyprotein and are proximally associated in preintegration complexes, functional interplay is possible (16)(17)(18). Some evidence suggesting that mutations in RT and IN may cooperatively produce cross-class drug resistance and/or compensatory fitness effects has been presented. For example, HIV-1 site-directed mutants with certain combinations of INSTI and NNRTI resistance substitutions, such as G140S/Q148H in IN with K103N in RT, Citation Andreatta KN, Goodman DD, Miller MD, White KL. 2015. Reduced viral fitness and lack of cross-class resistance with integrase strand transfer inhibitor and nucleoside reverse transcriptase inhi...

Section: Methodsmentioning

confidence: 99%

Reduced Viral Fitness and Lack of Cross-Class Resistance with Integrase Strand Transfer Inhibitor and Nucleoside Reverse Transcriptase Inhibitor Resistance Mutations

Andreatta

Goodman

Antimicrob Agents Chemother

et al. 2015

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“…Viral growth competitions were performed by coculturing competing recombinant xxLAI viruses in MT-2 cells for 9 days in the presence or absence of ARV inhibitors, as described previously (18,20,21). MultiCode RTx PCR (Luminex, Austin, TX) was performed on extracted viral RNA from days 0, 3, 6, and 9 as described previously with allele-specific forward and reverse primers (20,21,26). Percentages of competing viruses were determined from standard curves generated by SigmaPlot.…”

Section: Methodsmentioning

confidence: 99%

Drug Susceptibility and Viral Fitness of HIV-1 with Integrase Strand Transfer Inhibitor Resistance Substitution Q148R or N155H in Combination with Nucleoside/Nucleotide Reverse Transcriptase Inhibitor Resistance Substitutions

Andreatta

Antimicrob Agents Chemother

White

2016

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In clinical trials of coformulated elvitegravir (EVG), cobicistat (COBI), emtricitabine (FTC), and tenofovir disoproxil fumarate (TDF), emergent drug resistance predominantly involved the FTC resistance substitution M184V/I in reverse transcriptase (RT), with or without the tenofovir (TFV) resistance substitution K65R, accompanied by a primary EVG resistance substitution (E92Q, N155H, or Q148R) in integrase (IN). We previously reported that the RT-K65R, RT-M184V, and IN-E92Q substitutions lacked cross-class phenotypic resistance and replicative fitness compensation. As a follow-up, the in vitro characteristics of mutant HIV-1 containing RT-K65R and/or RT-M184V with IN-Q148R or IN-N155H were also evaluated, alone and in combination, for potential interactions. Single mutants displayed reduced susceptibility to their corresponding inhibitor classes, with no cross-class resistance. Viruses with IN-Q148R or IN-N155H exhibited reduced susceptibility to EVG (137-and 40-fold, respectively) that was not affected by the addition of RT-M184V or RT-K65R/M184V. All viruses containing RT-M184V were resistant to FTC (>1,000-fold). Mutants with RT-K65R had reduced susceptibility to TFV (3.3-to 3.6-fold). Without drugs present, the viral fitness of RT and/or IN mutants was diminished relative to that of the wild type in the following genotypic order: wild typeIn the presence of drug concentrations approaching physiologic levels, drug resistance counteracted replication defects, allowing single mutants to outcompete the wild type with one drug present and double mutants to outcompete single mutants with two drugs present. These results suggest that during antiretroviral treatment with multiple drugs, the development of viruses with combinations of resistance substitutions may be favored despite diminished viral fitness. E volution of multiple HIV mutations occurs during prolonged antiretroviral (ARV) treatment failure. These mutations typically can be categorized as polymorphic, resulting in no phenotypic change to the virus, or resistance associated, resulting in reduced susceptibility to one or more ARV inhibitors. Additional accessory mutations may also develop to produce combined effects on viral replicative fitness and/or drug susceptibility. Several such relationships between mutations within the same coding region of a target enzyme have been characterized; for example, the G140S substitution in integrase (IN-G140S) has been shown to restore viral fitness of the IN-Q148H substitution and enhance resistance to raltegravir (RAL), an integrase strand transfer inhibitor (INSTI) (1, 2). Additionally, substitutions in one coding region may exhibit effects on susceptibility to ARV inhibitors from a different drug class: for example, the addition of IN-G140S/ Q148R to a virus with the nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance substitution K103N appears to significantly enhance resistance to the NNRTI efavirenz (EFV) (3). While data on such cross-class interactions is limited (4, 5), reverse transcriptase (R...

“…After the optimal cDNA dilution was established, 95 reactions were amplified through 2 rounds of PCR to attain 20 to 30 (ϳ30%) clones. These clones were then purified, sequenced using Applied Biosystems BigDye Terminator chemistry, sequenced using Applied Biosystems genetic analyzer technology, and analyzed as previously described (21).…”

Section: Methodsmentioning

confidence: 99%

“…Clonal analyses and single-genome sequencing (SGS) provide a greater capacity to detect a minor population but are highly labor-intensive. A simpler allelespecific real-time PCR (AS-PCR) method using the Multicode RTx real-time PCR technology (EraGen Biosciences) has been reported to quantify HIV-1 reverse transcriptase mutants down to a level of 0.01% in plasmid DNA mixtures and to 0.5% in preamplified PCR products (14,20,21). Taking advantage of this technology, we have developed a highly sensitive AS-PCR assay to detect low-frequency NS5B Y448H mutant variants in laboratory and clinical samples.…”

mentioning

confidence: 99%

Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples

Bae

et al. 2011

J Clin Microbiol

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The Y448H mutation in NS5B has been selected by GS-9190 as well as several benzothiadiazine hepatitis C virus (HCV) polymerase inhibitors in vitro and in vivo. However, the level and the evolution kinetics of this resistance mutation prior to and during treatment are poorly understood. In this study, we developed an allele-specific real-time PCR (AS-PCR) assay capable of detecting Y448H when it was present at a level down to 0.5% within an HCV population of genotype 1a or 1b. No Y448H mutation was detected above the assay cutoff of 0.5% in genotype 1b-infected Con-1 replicons prior to in vitro treatment. However, the proportion of replicons with the Y448H mutation rapidly increased in a dose-dependent manner upon treatment with GS-9190. After 3 days of treatment, 1.2%, 6.8%, and >50% of the replicon population expressed Y448H with the use of GS-9190 at 1, 10, and 20 times its 50% effective concentration, respectively. In addition, plasma from 65 treatment-naïve HCV-infected patients (42 and 23 with genotype 1a and 1b, respectively) was tested for the presence of Y448H by AS-PCR and population sequencing. As expected, all patient samples were wild type at NS5B Y448 by population sequencing. AS-PCR results were obtained for 62/65 samples tested, with low levels of Y448H ranging from 0.5% to 3.0% detected in 5/62 (8%) treatment-naïve patient samples. These findings suggest the need for combination therapy with HCV-specific inhibitors to avoid viral rebound of preexisting mutant HCV.Hepatitis C virus (HCV) is a global health problem and infects more than 170 million individuals worldwide (15). The current standard treatment with pegylated interferon and ribavirin is complicated by frequent adverse reactions, and a sustained virologic response (SVR) can be achieved in only ϳ50% of patients infected with the most prevalent genotype, genotype 1. HCV NS5B polymerase is essential for viral replication, and a number of compounds that inhibit this enzyme (HCV polymerase inhibitors) have been discovered; some have advanced to phase I/II clinical trials and have demonstrated antiviral activity in HCV-infected patients in monotherapy (8). However, monotherapy with any specific HCV nonnucleoside inhibitors (NNIs) resulted in the rapid selection of resistance mutations in the replicon and HCV genotype 1-infected patients (19). Among the resistance mutations, Y448H in NS5B has been reported to be selected by GS-9190, as well as by the benzothiadiazine and acylpyrrolidine classes of NNIs (4, 13). The NS5B Y448H mutant remained sensitive to interferon, ribavirin, and inhibitors of HCV NS3 protease, NS5A, and NS5B (site II nonnucleoside and nucleoside) (4, 12). HCV has extremely high genetic diversity and exists in HCV-infected individuals as a pool of closely related but distinct variants as quasispecies. These viral quasispecies may include drug-resistant variants existing within a predominantly wild-type virus population before treatment (2, 5, 7). The presence of drug-resistant variants at different low-level frequencies in HCV...