Tambuyzer, Lotke MSc*; Nijs, Steven MSc*; Daems, Bjorn MSc*; Picchio, Gaston PhD†; Vingerhoets, Johan PhD*
Analyses of baseline resistance data from the DUET (TMC125 to Demonstrate Undetectable viral load in patients Experienced with ARV Therapy) studies have resulted in the identification of 17 etravirine resistance-associated mutations (RAMs) based on their association with decreased virologic response and/or increased etravirine fold change in 50% effective concentration (FC)1: V90I, A98G, L100I, K101E/H/P, V106I, E138A, V179D/F/T, Y181C/I/V, G190A/S, and M230L.
A recent analysis described the genotypic and phenotypic changes at endpoint relative to baseline, including the emergence of individual reverse transcriptase (RT) mutations, in patients who received the nonnucleoside reverse transcriptase inhibitor (NNRTI) etravirine in the phase III DUET studies and who experienced virologic failure by rebound.2 Mutations at positions E138 (n = 17), V179 (n = 35), and Y181 (n = 15) emerged most frequently in these patients. In contrast to several mutations at positions V179 and Y181 (V179D/F/T and Y181C/I/V), only E138A is included in the etravirine-weighted genotypic score.
Because there is an important structural and functional contribution to development of NNRTI resistance from E138 in the p51 subunit located at the edge of this pocket,3,4 the objective of the current analysis was to define the contribution of E138 variants to the development of etravirine resistance based on: 1) the emergence of E138 mutations in patients virologically failing on an etravirine-containing regimen in the DUET studies; 2) the baseline prevalence of E138 mutations and association with virologic response in etravirine-treated patients from the DUET studies; and 3) the phenotypic susceptibility data for etravirine generated using HIV-1 site-directed mutants (SDMs) harboring one of the E138 mutations. In addition, individual patient data are discussed to support the understanding of the role of E138 mutations in etravirine resistance.
MATERIALS AND METHODS
Genotyping and Phenotyping
Genotypic analyses were performed by automated population sequencing (Virco BVBA, Mechelen, Belgium). Individual data were reported as amino acid changes along the HIV-1 protease (PR) and RT genes as compared with the HIV-1/HXB2 wild-type reference.5 An emerging mutation was defined as a mutation that could be detected at endpoint, but not at baseline.
For phenotypic analyses, recombinant clinical isolates were constructed according to the Antivirogram method (Virco BVBA).5,6 Briefly, PR and RT coding sequences were amplified from patient-derived viral RNA with HIV-1-specific primers. After homologous recombination of amplicons into a PR-RT-deleted proviral clone, the resulting recombinant viruses were harvested and used for in vitro sensitivity testing.
To assess phenotypic susceptibility to etravirine in vitro in the presence of specific E138 mutations, HIV-1 SDMs each harboring one of the various E138 mutations were constructed in a pGEM(r) vector encompassing the PR and RT coding sequence of HIV-1/HXB2 using the QuikChange SDM kit (Stratagene, La Jolla, CA) and tested by the Antivirogram method.5-7
Clinical and Nonclinical Data Used for Analyses
The emergence of E138 mutations at endpoint was analyzed in pooled data from the DUET studies8 for patients who initiated treatment with 200 mg etravirine twice daily in combination with a background regimen of darunavir/ritonavir, investigator-selected nucleoside/nucleotide reverse transcriptase inhibitors, and optional enfuvirtide, and who experienced virologic failure by rebound between baseline and Week 96 (n = 93; referred to as rebounders). Virologic failure by rebound was defined as two consecutive measurements of plasma viral load 0.5 log10 or greater HIV-1 RNA copies/mL above the nadir after a confirmed virologic response (two consecutive measurements of 1 log10 or greater HIV-1 RNA copies/mL below baseline).
At the time of analysis, all patients had received 96 weeks of treatment or had discontinued earlier. Patients were included if they had genotypic and phenotypic data available at endpoint, defined as the last visit within the treatment phase (starting from the first intake day of etravirine until either the Week 96 cutoff date or until 7 days after the last day of etravirine intake).
The prevalence and effect of E138 mutations on virologic response to etravirine (less than 50 HIV-1 RNA copies/mL) was studied in etravirine-treated DUET patients not using enfuvirtide de novo and excluding those who discontinued for other reasons than virologic failure (n = 406). For comparison, the overall prevalence of E138 mutations was further assessed in a broader data set containing genotypes and phenotypes for 4248 NNRTI-resistant HIV-1 recombinant clinical isolates.
Assessment of Etravirine Resistance Based on Genotype and/or Phenotype
To assess genotypic susceptibility to etravirine, the previously identified etravirine-weighted genotypic score (0-2, 2.5-3.5, and 4 or greater)1 was determined. This score is based on the sum of the weight factors of 17 individual etravirine RAMs present at baseline, ie, V90I, A98G, L100I, K101E/H/P, V106I, E138A, V179D/F/T, Y181C/I/V, G190A/S, and M230L.1
A list of previously identified NNRTI RAMs was taken into account in the current analysis: V90I, A98G, L100I, K101E/H/P/Q, K103H/N/S/T, V106A/I/M, V108I, E138A/G/K/Q, V179D/E/F/G/I/T, Y181C/I/V, Y188C/H/L, V189I, G190A/C/E/Q/S, H221Y, P225H, F227C/L, M230I/L, P236L, K238N/T, and Y318F.1,9
To assess phenotypic susceptibility to etravirine, viruses originating from HIV-1-infected patients or HIV-1 SDMs were considered to be susceptible or resistant based on the previously defined etravirine clinical cutoffs (FC ≤ 3, > 3-≤ 13, or > 13).1
Emergence of Mutations at Position E138
Analysis of the 93 rebounders who had been treated with etravirine for up to 96 weeks and had available genotype and phenotype data at endpoint demonstrated that 15 patients (16%) showed an emerging E138 mutation (single or mixture) at endpoint. All 15 patients harbored HIV-1 subtype B. The observed amino acids at position E138 at endpoint were G (n = 5), Q (n = 5), K (n = 3), V (n = 2), P (n = 1), and A (n = 1).
For each individual patient, the evolution of the genotypic and phenotypic determinants from baseline to endpoint during treatment with etravirine is shown in Table 1. The overall median etravirine FC for the 15 rebounders was 2.2 at baseline and 47.4 at endpoint. In 14 of 15 patients, etravirine RAMs were already present at baseline or coemerged with the E138 mutation at endpoint (Table 1). The most frequently observed NNRTI RAMs (n ≥ 2) at baseline were K103N (n = 5), V179I (n = 4), V90I (n = 3), Y181C (n = 3), V189I (n = 3), G190A (n = 3), A98G (n = 2), L100I (n = 2), and Y188L (n = 2). The most frequently emerging NNRTI RAMs (n ≥ 2) at endpoint were V179I (n = 4), Y181C (n = 4), K101E (n = 2), V108I (n = 2), V179F (n = 2), and V189I (n = 2), as described previously.2
In all patients with an emerging E138 mutation at endpoint, an associated increase in etravirine FC above the preliminary upper clinical cutoff of etravirine (greater than 13) was observed. For one patient with an emerging E138G mutation at endpoint that developed from an E138A mutation at baseline (Patient E in Table 1), the etravirine FC was already greater than 13 (FC = 184.3) at baseline as a result of the presence of Y181I and increased further at endpoint (FC = 1135). This patient was one of the six patients with an etravirine-weighted genotypic score of 2.5 or greater at baseline (see Table 1; 4.5 is the sum of weight factors 1.5 and 3 for E138A and Y181I, respectively), predicting an intermediate virologic response to etravirine. For the nine remaining patients with a predicted highest response to etravirine at baseline, seven developed additional etravirine RAMs resulting in an etravirine-weighted genotypic score of 2.5 or greater.
An increase in etravirine FC above the preliminary upper clinical cutoff of 13 at endpoint caused by the emergence of an E138 mutation alone (without the other NNRTI RAMs or etravirine RAMs) was observed in three rebounders, two with emerging E138K and one with emerging E138G. In one patient, decreased susceptibility for etravirine at endpoint was observed together with the emergence of a mixture of E138A/P/Q, in which E138A was previously identified as an etravirine RAM.
For the four rebounders with emerging E138Q, either the NNRTI RAMs V179I and Y181C (with K101E for 1 rebounder) (n = 3) or the etravirine RAM K101E (n = 1) emerged together with E138Q. For the three rebounders with emerging E138K, either L100I was already present at baseline (n = 2) or L100I emerged together with E138K (n = 1).
Prevalence of E138 Mutations and Their Effect on Virologic Response to Etravirine
In the subset of 406 etravirine-treated DUET patients not using enfuvirtide de novo and excluding those who discontinued for other reasons than virologic failure, only E138A and E138Q were observed at baseline. E138A and E138Q were observed in 3% (12 of 406) and 2.5% (10 of 406) of patients at baseline, respectively. The presence of E138A was associated with a response rate of 50% (six of 12 patients achieved less than 50 HIV-1 RNA copies/mL) and the presence of E138Q with a response rate of 80% (eight of 10 patients). In the total intent-to-treat population of the DUET studies (n = 1203), E138G was present at baseline in two patients, both of whom responded to etravirine treatment. The overall prevalence of E138 mutations was also low when assessed in a large panel of recombinant clinical isolates resistant to efavirenz or nevirapine (n = 4248): 2.5% for E138A, 1.1% for E138Q, 0.7% for E138G, 0.6% for E138K, 0.05% for E138S, 0.02% for E138V, and 0% for both E138P and E138R.
Phenotypic Susceptibility Against Etravirine of Site-Directed Mutants Harboring E138 Mutations
SDMs harboring the single E138 mutations A, G, K, Q, R, and S were constructed and tested for their phenotypic susceptibility to etravirine. FC values are presented in Table 2. FC measurements for the six SDMs ranged between 2.4 and 3.6. SDMs harboring single etravirine RAMs for which a weight factor less than 2.5 was assigned in the etravirine-weighted genotypic score (n = 17) had similar etravirine FC values ranging from less than 0.2 to 2.9.1,7
Addition of E138 Mutations in the Etravirine-Weighted Genotypic Score Based on Genotypic and Phenotypic Observations
The mutations E138G, E138K, and E138Q are added to the etravirine-weighted genotypic score based on their emergence in more than one of the DUET rebounders associated with a decrease in etravirine susceptibility combined with a prevalence higher than 0.5% in NNRTI-resistant clinical isolates. Consequently, the list of etravirine RAMs now includes 20 RAMs. Because their effect on etravirine susceptibility in single SDMs was limited, and the effect on virologic response could only be assessed for E138Q, the relative weight factor for these E138 mutations was set at 1 (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A178, which shows the 2010 list of etravirine RAMs).
In Table 1, Patient E has an etravirine-weighted genotypic score of 3 at endpoint (weight factor 3 for Y181I), which becomes 4 using the new score (addition of weight factor 1 for E138G). For Patient L, the endpoint score of 2.5 (L100I alone) becomes 3.5 (addition of E138K). In both cases, the concordance of the etravirine-weighted genotypic score with the etravirine FC improves.
In this study, the contribution of E138 mutations to decreased susceptibility to etravirine was investigated after a recent analysis reporting frequent changes at this amino acid position in RT in etravirine-treated patients.2 Fifteen patients (16%) with a change at position E138 (including G, Q, K, V, P, and A) among the emerging mutations at endpoint were identified in the 93 rebounders from the pooled DUET studies. In all 15 patients, decreased susceptibility to etravirine (FC greater than 13) was observed at endpoint. In all but two patients, the emergence of E138 mutations was accompanied by at least one etravirine RAM present either already at baseline or emerging together at endpoint, suggesting that the decreased susceptibility to etravirine was likely not caused solely by the E138 mutation but rather by a combined effect of E138 and etravirine RAMs. This observation was confirmed by the low etravirine FC values observed in SDMs harboring single E138 mutations.
The effect of mutations at position E138 on NNRTI resistance in vitro was first described for the 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole-2'',2''-dioxide) derivatives.10-15 Resistance was caused mainly by E138K, but also by E138Q and E138G. Recent in vitro data suggested that viral clones containing E138K displayed low-level phenotypic resistance to etravirine and modestly impaired replication capacity (twofold) compared with wild-type virus.16 E138 did not appear to cause high-level resistance to efavirenz or nevirapine.16 Furthermore, E138K was shown to play a role in resistance to the novel NNRTI IDX899 and its emergence was observed in selection experiments with etravirine.16,17 In addition, pooled results from the Phase III Efficacy Comparison in Treatment Naive HIV-Infected Subjects Of tmc278 and Efavirenz (ECHO) and Tmc278 Against HIV, In A Once-Daily Regimen Versus Efavirenz (THRIVE) trials show that the most frequently emerging NNRTI RAM in TMC278-treated patients was E138K.18
Previous studies suggested a significant impact of E138G and E138Q on etravirine resistance demonstrated by correlation with phenotype in clinical samples.19,20 The effect of E138G and E138Q, but also E138A and E138K, on etravirine susceptibility was confirmed in a large matched genotype-phenotype database.21 In the DUET data set, the virologic response in the presence of E138G or E138K could not be determined, but emergence of these mutations in patients who failed during etravirine treatment was associated with an increased etravirine FC in all cases.
The list of 17 etravirine RAMs was updated to include E138G, E138K, and E138Q in the etravirine-weighted genotypic score, resulting in a total list of 20 RAMs. The relative weight factor for these E138 mutations was set at 1. Single E138 mutations are associated with low resistance, and not complete loss of susceptibility, to etravirine.
The authors thank the patients and their families, the study coordinators, and the investigators who participated in the clinical studies. The authors acknowledge Karen Runcie (Medical Writer, Gardiner-Caldwell Communications, Macclesfield, UK) for providing medical writing support and collating author contributions.
1. Vingerhoets J, Tambuyzer L, Azijn H, et al. Resistance profile of etravirine: combined analysis of baseline genotypic and phenotypic data from the DUET studies. AIDS
2. Tambuyzer L, Vingerhoets J, Azijn H, et al. Characterization of genotypic and phenotypic changes in HIV-1-infected patients with virologic failure on an etravirine-containing regimen in the DUET-1 and DUET-2 clinical studies. AIDS Res Hum Retroviruses
3. Kohlstaedt LA, Wang J, Friedman JM, et al. Crystal structure at 3.5 A° resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science
4. de Bethune MP. Non-nucleoside reverse transcriptase inhibitors (NNRTIs), their discovery, development, and use in the treatment of HIV-1 infection: a review of the last 20 years (1989-2009). Antiviral Res
5. Hertogs K, de Béthune MP, Miller V, et al. A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs. Antimicrob Agents Chemother
6. Pauwels R, Hertogs K, Kemp S, et al. Comprehensive HIV drug resistance monitoring using rapid, high-throughput phenotypic and genotypic assays with correlative data analysis. Presented at the 2nd International Workshop HIV Drug Resistance; 1998; Lake Maggiore, Italy. Abstract 51.
7. Azijn H, Tirry I, Vingerhoets J, et al. TMC278, a next-generation NNRTI, active against wild-type and NNRTI-resistant HIV-1. Antimicrob Agents Chemother
8. Katlama C, Clotet B, Mills A, et al. Efficacy and safety of etravirine at Week 96 in treatment-experienced HIV-1-infected patients in the DUET-1 and DUET-2 trials. Antivir Ther
9. Tambuyzer L, Azijn H, Rimsky LT, et al. Compilation and prevalence of mutations associated with resistance to non-nucleoside reverse transcriptase inhibitors. Antivir Ther
10. Pelemans H, Aertsen A, Van Laethem K, et al. Site-directed mutagenesis of human immunodeficiency virus type 1 reverse transcriptase at amino acid position 138. Virology
11. Jonckheere H, Taymans JM, Balzarini J, et al. Resistance of HIV-1 reverse transcriptase against [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)] (TSAO) derivatives is determined by the mutation Glu138 -->Lys on the p51 subunit. J Biol Chem
12. Balzarini J, Karlsson A, Vandamme A-M, et al. Human immunodeficiency virus type 1 (HIV-1) strains selected for resistance against the HIV-1-specific [2',5'-bis-O-(tertbutyldimethylsilyl)-3'-spiro-5”-(4”-amino-1”,2”-oxathiole-2”,2”-dioxide)]-f8-D-pentofuranosyl (TSAO) nucleoside analogues retain sensitivity to HIV-1-specific nonnucleoside inhibitors. Proc Natl Acad Sci U S A
13. Balzarini J, Karlsson A, Pérez-Pérez MJ, et al. Treatment of human immunodeficiency virus type 1(HIV-1)-infected cells with combinations of HIV-1-specific inhibitors results in a different resistance pattern than does treatment with single-drug therapy. J Virol
14. Balzarini J, Karlsson A, Sardana VV, et al. Human immunodeficiency virus 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors may suppress the replication of specific drug-resistant (E138K) RT HIV-1 mutants or select for highly resistant (Y181C -* C1811) RT HIV-1 mutants. Proc Natl Acad Sci U S A
15. Balzarini J. Suppression of resistance to drugs targeted to human immunodeficiency virus reverse transcriptase by combination therapy. Biochem Pharmacol
16. Asahchop E, Oliveira M, Wainberg M, et al. Characterization of the E138K resistance mutation in HIV-1 reverse transcriptase conferring susceptibility to etravirine in B and non-B HiV-1 subtypes. Antimicrob Agents Chemother
17. Jakubik JJ, Chapron C, Hubbard L, et al. In vitro cross-resistance profile for a next-generation NNRTI: IDX899. Antiviral Ther
. 2008;13(suppl 3):A28.
18. Rimsky L, Eron J, Clotet B, et al. Characterization of the resistance profile of TMC278: 48-week analysis of the Phase III studies ECHO and THRIVE. Presented at the 50th Interscience Conference on Antimicrobial Agents and Chemotherapy; 2010; Boston, MA. Abstract H-1810.
19. Kagan RM, Sista P, Pattery T, et al. Additional HIV-1 mutation patterns associated with reduced phenotypic susceptibility to etravirine in clinical samples. AIDS
20. Winters B, Villacian J, Van Craenenbroeck E, et al. Development of virco(r) type HIV-1 resistance analysis, including clinical cutoffs for TMC125 (etravirine, ETR), a new NNRTI. Presented at the 15th Conference on Retroviruses and Opportunistic Infections; 2008; Boston, MA. Abstract 873.
21. Haddad M, Stawiski E, Benhamida J, et al. Improved genotypic algorithm for predicting etravirine susceptibility: comprehensive list of mutations identified through correlation with matched phenotype. Presented at the 17th Conference on Retroviruses and Opportunistic Infections; 2010; San Francisco, CA. Abstract 574.
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