Distinct resistance patterns to etravirine and rilpivirine in viruses containing nonnucleoside reverse transcriptase inhibitor mutations at baseline
Asahchop, Eugene L.a,b; Wainberg, Mark A.a; Oliveira, Maureena; Xu, Hongtaoa; Brenner, Bluma G.a; Moisi, Danielaa; Ibanescu, Ilinca R.a; Tremblay, Cecileb,c
aMcGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital
bCentre de Recherche du Centre Hospitalier de I’Université de Montréal
cLaboratoire de Santé Publique du Québec, Institut National de Santé Publique du Québec, Montréal, Québec, Canada.
Correspondence to Mark A. Wainberg, 3755 Cote–Ste-Catherine-Road, Montreal, QC H3T1E2, Canada. Tel: +1 514 340 8260; fax: +1 514 340 7537; e-mail: email@example.com
Received 16 August, 2012
Revised 28 November, 2012
Accepted 6 December, 2012
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.AIDSonline.com).
Objective: The current in-vitro study examined HIV-1 drug resistance patterns following etravirine (ETR) and rilpivirine (RPV) drug pressure in viruses containing baseline nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations.
Design and method: Several baseline mutations were introduced into NL-4.3 (subtype B clone) by site-directed mutagenesis. This virus, together with two subtype C clinical isolates containing baseline mutations, was passaged in increasing drug pressure of NNRTIs in cord blood mononuclear cells. Genotypic analysis was performed at different weeks. Phenotypic resistance for ETR, RPV, and efavirenz (EFV) and the replication capacity of several mutations and combinations were tested.
Results: In wild-type viruses and viruses containing K103N alone at baseline, E138K or E138G mutations were observed following pressure with either ETR or RPV prior to the appearance of other NNRTI resistance mutations. These changes were observed regardless of viral subtype. However, subtype B viruses containing Y181C generated V179I/F or A62V/A but not E138K following exposure to ETR or RPV, respectively, whereas subtype C viruses containing Y181C developed E138V together with Y188H and V179I under ETR pressure. The addition of mutations at position 138 to Y181C did not significantly enhance levels of resistance to ETR or RPV. The replicative capacity of viruses containing Y181C and either E138K or E138A was similar to that of viruses containing either E138K or E138A alone.
Conclusion: These results demonstrate that ETR and RPV are likely to select for E138K as a major resistance mutation if no or very few other resistance mutations are present and that Y181C may be antagonistic to E138K.
Antiretroviral therapy (ART) has significantly decreased HIV-associated morbidity and mortality . Nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) are two classes of drugs that are used clinically ; the latter are noncompetitive inhibitors of HIV-1 reverse transcriptase that bind to a hydrophobic pocket in the p66 subunit near the polymerase active site, allosterically inhibiting the activity of the enzyme . NNRTIs are routinely prescribed for both treatment-naive and treatment-experienced patients [3–5]. However, nonadherence to therapy can result in the selection of drug-resistance mutations [6,7].
Multiple studies have reported an increasing prevalence of NNRTI drug resistance mutations in acute HIV-1 infection [8,9]. Individuals harboring at least one transmitted NNRTI resistance mutation are less likely to respond positively to treatment regimens that include first-generation NNRTIs such as nevirapine (NVP) and efavirenz (EFV) [10,11]. The widespread use of single dose NVP to prevent mother-to-child transmission of HIV-1 has increased the prevalence of NNRTI resistance mutations (reviewed in ). In addition, cross-resistance among NNRTI is common , such that the sequential use of first generation NNRTIs is excluded in treatment-experienced patients. Etravirine (ETR) is a second generation NNRTI that possesses high activity against both wild-type and drug-resistant viruses at nmol/l concentrations and was approved for use in treatment-experienced patients with NNRTI-resistant viruses [14,15]. ETR can bind to reverse transcriptase by rotating within the NNRTI binding pocket to accommodate resistance mutations and retain activity  and has been shown to be effective in treatment-experienced, NNRTI-resistant patients in the DUET-1 and DUET-2 studies (Demonstrate undetectable viral load in patients experienced with antiretroviral therapy) [5,17]. These same studies identified 20 ETR resistance-associated mutations (RAMs) that can diminish virological response: V90I, A98G, L100I, K101E/H/P, V106I, E138A/K/G/Q, V179D/F/T, Y181C/I/V, G190A/S, and M230L [18,19]. Each of these mutations has been assigned a weighted genotypic score and three or more of these mutations are required to significantly reduce virologic response to ETR .
Rilpivirine (RPV) is another second generation NNRTI whose mode of binding within the NNRTI binding pocket is similar to that of ETR . In recent phase III studies [ECHO (Efficacy comparison in treatment-naïve HIV-infected subjects of TMC-278 and efavirenz) and THRIVE (TMC-278 against HIV in a once-daily regimen vs efavirenz)], HIV-1 drug-naive patients who failed combination therapy containing RPV and emtricitabine (FTC) or lamivudine (3TC) frequently harbored the resistance mutations E138K/M184I and E138K/M184V [21,22]. The same mutations are also selected in culture by RPV and ETR, resulting in high-level cross-resistance [23,24].
The most prevalent NNRTI mutations for EFV and NVP include K103N, Y181C and G190A , of which K103N does not compromise susceptibility to either ETR or RPV [14,23]. However, both Y181C and G190A can affect responsiveness to ETR  while only Y181C decreases susceptibility to RPV . In contrast, the application of ETR pressure against wild-type viruses commonly selects for the E138K mutation that has also been identified in patients who have failed first-line regimens that have included RPV.
The current study was performed to determine the basis for the selection of NNRTI mutations other than E138K in the DUET clinical studies in which patients had received a NNRTI as part of first-line therapy. We also investigated whether HIV-1 that already contained E138K or M184I/V or E138K and M184I/V could develop additional resistance mutations in the presence of ETR or RPV.
Materials and methods
Viral isolates, cells, drugs, and plasmids
The subtype C viral isolates 10680 and 4743 were obtained with informed consent from HIV-1-infected patients at our clinics in Montreal, Canada. Cord blood mononuclear cells (CBMCs) were obtained through the Department of Obstetrics, Jewish General Hospital, Montreal, Canada. The HEK293T cell line was obtained from the American Type Culture Collection. The infectious molecular clone pNL-4.3 and TZM-bl cells were obtained through the NIH AIDS Research and Reference Reagent Program, courtesy of Malcolm Martin and John C. Kappes, respectively. ETR and dapivirine (TMC120) were provided by Janssen Inc., whereas RPV and EFV were obtained from the NIH AIDS Research and Reference Reagent Program. Tenofovir (TFV), also used in selections, was provided by Gilead Sciences, Inc., Foster City, California, USA.
Site-directed mutagenesis and virus production
Amino acid changes in reverse transcriptase were introduced into the pNL-4.3 plasmid by site-directed mutagenesis (SDM) using a QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, California, USA). Mutations were introduced individually by PCR in the case of double mutants. The list of mutations and primers employed are shown in a table (supplemental digital content 1, http://links.lww.com/QAD/A295). The presence of mutations was confirmed by sequencing, and DNA was ultimately transformed into DH5α cells (Invitrogen, Montreal, Canada) for high yield of plasmid. Viruses were produced by transfection as described previously . Virus production was confirmed by measurement of reverse transcriptase activity and p24 production.
Selection of resistance mutations in cord blood mononuclear cells
Phytohemagglutinin-stimulated CBMCs were infected with viruses (multiplicity of infection of 0.1) for 2 h, incubated at 37°C, and subsequently washed with RPMI 1640 media (Invitrogen), supplemented with 10% fetal bovine serum, and seeded into a 24-well plate at a density of 2.5 × 106 cells per well . Selection for resistance in CBMCs was performed using increasing concentrations of drugs (ETR and EFV) at concentrations starting below the 50% effective concentration (EC50) of the drugs . As controls, all viruses were simultaneously passaged in the absence of drugs. Reverse transcriptase assays were performed weekly as described to monitor viral replication [29,30]. On the basis of the ratio of reverse transcriptase values in culture fluids of control wells/wells with drug at the previous round of replication, drug concentrations were increased at subsequent passages. Selection at a particular drug concentration was considered to be complete when repeated passage revealed that reverse transcriptase levels in culture fluids had peaked at the same time as that of a control well that did not contain drugs. Virus-containing culture fluids were harvested and kept at −80°C for subsequent genotypic analysis at the same time that drug concentrations were increased. Selections for resistance were performed over 25–30 weeks.
Nucleic acid extraction, amplification, and sequencing analysis
Viral RNA was extracted from culture supernatants using the Qiagen QIAamp viral extraction kit (Mississauga, Ontario, Canada). Viral RNA amplification was performed by reverse transcriptase-PCR (RT-PCR) and nested PCR using a previously published protocol (Virco BVBA, Mechelen, Belgium). The resulting PCR-amplified DNA fragment was purified using the QIAquick PCR purification kit (Mississauga, Ontario, Canada), as specified by the manufacturer. The presence of the 1.5 kb protease and RT-PCR products was confirmed by running 5 μl of each product on a 1% agarose gel with Sybersafe (Invitrogen).
Genotyping was performed by a published protocol (Virco BVBA), based on sequencing of the first 400 amino acids in the reverse transcriptase region as described . Data were analyzed using SeqScape software version 2.5.
Phenotypic drug susceptibility in TZM-bl cells
Drug susceptibility to NNRTIs (ETR, RPV, TMC120, and EFV) was measured in a single cycle cell-culture-based phenotypic assay in TZM-bl cells. In brief, 20 000 cells per well were added into a 96-well culture plate in 50 μl of Dulbecco modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Mississauga, Toronto, Canada), 1% penicillin-streptomycin, and 1% L-glutamine (Invitrogen). Cells were infected with either wild-type or mutant HIV and serially diluted drugs were added. Standardization of virus infections was determined by adding virus at a p24 concentration that would yield approximately 160 000 ± 20 000 relative light units (RLU) as detected by luminescence. All cultures were maintained at 37°C under 5% CO2 for 48 h. After 48 h, 100 μl of Bright-Glo reagent (Promega, San Francisco, California, USA) was added to 100 μl of infected TZM-bl cells. Drug efficacy was determined by quantifying luciferase activity as a measure of viral replication. RLU were detected with a 1450 MicroBeta TriLux microplate scintillation and luminescence counter (Perkin-Elmer). The EC50 values were determined by nonlinear regression with GraphPad Prism (version 5.01) software. Resistance to a given drug was determined based on the previously published lower clinical cutoff (CCO) of a drug which determines the cutoff fold change value for drug sensitivity. These values are 3.0 for ETR, 3.4 for EFV and 2.0 for RPV [18,31,32].
Determination of relative replication capacity in TZM-bl cells
The replicative capacities of competent clonal wild-type and mutant HIV-1 were evaluated in a noncompetitive infectivity assay using TZM-bl cells . Twenty thousand cells per well were added into a 96-well culture plate in 100 μl of DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin, and 1% L-glutamine (Invitrogen). Viral stocks for both wild-type and mutant viruses were normalized by p24 and recombinant viruses were serially diluted two-fold from viral stock suspensions. After 4 h, 50 μl of DMEM were removed from the wells and replaced by 50 μl of virus dilution; a control well did not contain virus. Virus and cells were co-cultured for 48 h, after which 100 μl of Bright-Glo reagent was added and luciferase activity was measured in a luminometer as described (Promega). Viral replication levels were expressed as a percentage of RLU with reference to wild-type virus for each viral variant studied.
Mutations selected in the presence of rilpivirine drug pressure
To determine the impact of baseline NNRTI mutations on the pattern of RPV resistance, the following NNRTI mutations and combinations were introduced into wild-type NL-4.3 by SDM: K103N, G190A, Y181C, E138K, and M184I/V (Table 1). All viral clones were passaged in the absence of drugs and genotyped at the end of each passage. Genotyping results revealed that all baseline mutations were maintained at the end of the selections, except for viral clones containing the mutations M184I, M184V, and E138K/M184I, in which case, the M184I/V mutations reverted to wild-type.
In the wild-type virus, a combination of E138K and L100I was selected after 8 weeks in the presence of RPV (Fig. 1). Continuous RPV drug pressure resulted in the emergence of K101E as a predominant viral variant, whereas viruses containing the E138K and L100I mutations, previously selected in the dominant viral population, reverted to wild-type (Fig. 1). At week 21, the E138K and L100I mutations re-emerged and predominated at week 26, whereas K101E reverted to wild-type. In the virus containing Y181C at baseline, only the NRTI multidrug-resistant mutation A62A/V was selected, even when a RPV concentration of 300 nmol/l was attained. In viruses containing both K103N and G190A at baseline, E138K was the first mutation to emerge at week 12, whereas L100I was identified in the virus containing E138K at baseline (Table 1). In the viral clones containing E138K/M184I and M184V, no additional mutations developed. In contrast, clonal viruses containing E138K/M184V or M184I at baseline yielded H221H/Y or M41L and I135M, respectively, under RPV pressure.
Mutations selected in the presence of etravirine and etravirine/tenofovir drug pressure
The viral clones containing the mutations K103N, E138K, Y181C, M184I/V, G190A, K103N/Y181C, K103N/G190A, E138K/M184I, and E138K/M184V as well as two subtype C clinical isolates (isolates 4743 and 10680) containing baseline NNRTI mutations were exposed to ETR or combination of ETR and TFV for 20–30 weeks (Table 2). Beginning with suboptimal concentrations of ETR for 20–30 weeks in selection experiments, we observed distinct mutational pathways. In the subtype B (NL-4.3) wild-type clone and the subtype C wild-type clinical isolate, E138K/G was observed in all selections followed by other ETR RAMs. In contrast, subtype B viruses containing either K103N or G190A at baseline generated E138K/L100I and V106I, respectively, whereas subtype C viruses containing G190A developed E138K (Table 2). The clones containing either M184I or M184V yielded E138K, whereas the E138K/M184I and E138K/M184V clones developed the V189I and V118I mutations, respectively. Subtype B viruses containing Y181C at baseline commonly developed V179I/F but not E138 substitutions.
The subtype C clinical isolate containing Y181C as a baseline mutation developed E138V under ETR pressure together with Y188H and V179I; K238K/N and E399G were also frequently observed. When the combination of K103N/G190A was present in NL-4.3 (subtype B) at baseline, no mutation was selected, probably because the virus containing these mutations was not fit and was difficult to replicate in the presence of suboptimal drug concentration. Subtype C clinical isolates subjected to ETR/TFV drug pressure developed E138 mutations but not the TFV-associated K65R mutation (Table 2).
Mutations selected in the presence of efavirenz and efavirenz/tenofovir drug pressure
In the case of the subtype B (NL-4.3) wild-type and Y181C-containing viruses, the mutations selected under EFV pressure were K101E/Q, K103N, or Y188H. Viruses containing K103N generated the minor NNRTI mutations Y318F and N348I/N (see table, supplemental digital content 2, http://links.lww.com/QAD/A295). The subtype B NL-4.3 virus containing G190A at baseline mostly developed V106A. However, viruses containing the combinations of K103N/Y181C and K103N/G190A did not select additional substitutions. In the presence of M184I and M184V at baseline, amino acid substitutions at position 188 were observed, whereas viruses containing E138K/M184I or E138K/M184V developed G190A or E399G, respectively, under EFV drug pressure. In the clone containing E138K alone at baseline, the additional K103N and L100I mutations were observed (see table, supplemental digital content 2, http://links.lww.com/QAD/A295).
Subtype C viruses that were wild-type or that contained G190A or Y181C at baseline selected V106M, followed by the accumulation of several additional mutations (see table, supplemental digital content 2, http://links.lww.com/QAD/A295). The combination of EFV/TFV drug pressure did not select for K65R.
Mutations selected in the presence of dapivirine (TMC120) drug pressure
We also wanted to determine whether any additional mutations would develop in the presence of baseline mutations E138K, M184I, M184V, E138K/M184I, and E138K/M184V under dapivirine (TMC120) drug pressure. The latter is an NNRTI that is structurally related to RPV and ETR and that is now undergoing evaluation as an anti-HIV microbicide. After 20 weeks, wild-type viruses and viruses containing M184I and M184V yielded E138K, whereas viruses containing E138K/M184I or E138K/M184V developed L100I and V108I, respectively (see table, supplemental digital content 3, http://links.lww.com/QAD/A295).
In-vitro antiviral activity of nonnucleoside reverse transcriptase inhibitors against mutated viruses
The activity of the NNRTIs ETR, RPV, TMC120, and EFV were tested against NL-4.3 clones engineered to contain E138 and Y181C mutational combinations. Table 3 shows that the E138A and E138K amino acid substitutions conferred low-level resistance to ETR, RPV, and TMC120, whereas E138V, that was co-selected with Y181C in tissue culture, did not. Y181C alone conferred a fold-change in susceptibility of 6.0, 2.3, 5.3, and 2.5 for ETR, RPV, TMC120, and EFV, respectively. The addition of mutations at position 138 to Y181C did not significantly enhance levels of resistance to ETR, RPV, or TMC120 (Table 3). All of the combinations tested remained susceptible to EFV.
Impact of mutations at position 138 alone or in combination with Y181C on viral replication capacity
As E138K was not selected by ETR and RPV, when using virus that contained Y181C at baseline, we next wished to determine the viral replication capacity of viruses containing E138K and Y181C or E138 together with other mutations. For this purpose, TZM-bl cells were infected with serially diluted viral stocks (normalized for p24 levels and RLU) of wild-type virus or viruses containing the E138K, E138A, Y181C, E138K/Y181C, or E138A/Y181C mutations. The infectiousness of viral clones was determined by measuring luciferase activity at 48 h postinfection. The results show that the replication capacity of both the E138K and E138A viruses were decreased by approximately three-fold compared to wild-type, whereas that of Y181C virus was only slightly decreased by approximately 1.5-fold. The addition of E138K or E138A to Y181C further decreased replication capacity to about the same level (≈three-fold) as that of viruses containing either E138K or E138A alone (Fig. 2).
The current in-vitro study examined HIV resistance patterns following selection with ETR in viruses containing a variety of NNRTI mutations at baseline as well as in wild-type viruses. We showed that E138K or E138G was selected in each of a subtype B wild-type or K103N virus, a subtype C wild-type clinical isolate, and a subtype C virus containing G190A at baseline. However, subtype B viruses containing Y181C at baseline selected for V179I or V179F, but not E138K. These findings are consistent with previous in-vitro data that showed selection of E138K by ETR in wild-type viruses of multiple subtypes over 18 weeks . In the same study, it was shown that E138K emerged first and that Y181C, together with V179I/F, was selected subsequently due to increased ETR pressure . Thus, the present study shows that Y181C is antagonistic to E138K. When E138K is selected in wild-type viruses under ETR or RPV pressure, it is possible for Y181C and mutations at position 179 to be simultaneously selected, due to the fact that these additional mutations can enhance resistance levels over those obtained with compared E138K alone. Possibly, the use of ETR in drug-naive patients might also select for E138K, if such individuals were to experience virologic failure. Others have also observed differences in mutational patterns among viral subtypes in SUPT1 cells exposed to NNRTI pressure .
E138K was observed following RPV drug pressure in wild-type subtype B viruses and viruses containing either K103N or G190A at baseline, whereas subtype B viruses containing Y181C at baseline did not yield E138K. This observation is in agreement with the recent phase III trials (ECHO and THRIVE) in which RPV was shown to preferentially select for E138K in patients undergoing first-line treatment failure [21,22]. Under RPV drug pressure, wild-type viruses developed a mixture E138K, K101, and E101, that was dominated by E138K at the end of the selection experiment. This could be due to the presence of quasispecies that existed in the viral population and suggests that K101E and E138K cannot exist in the same viral clone possibly due to a salt bridge that exists between these two amino acids . Viral clones containing M184I/V at baseline did not select for NNRTI mutations after exposure to RPV, as observed with ETR in a recent in-vitro study . This could be due to decreased viral fitness of both the clones and difficulties in increasing RPV drug concentration. Our data are consistent with those from a previous study, in which it was shown that M184V viruses were less able to generate mutations under selection pressure and to escape from neutralizing antibodies on the basis of mutations in envelope than wild-type viruses .
In the DUET studies that led to the approval of ETR for use in NNRTI-experienced patients, only three subtype B patients who previously harbored K103N developed E138K and failed ETR therapy, whereas one patient who harbored Y181C developed E138V . However, most ETR-related mutations were at positions 138, 179, and 181 . A different study showed that 12 of 42 ETR failures who harbored mutations at position 181 at baseline contained at least one new NNRTI mutation but not E138K . In an in-vitro selection study, using a drug of the same family (TMC120), E138K was selected in wild-type virus but not in viruses containing Y181C at baseline . The present study, together with evidence from other studies [38,40], suggests that Y181C may be antagonistic to E138K. As shown here, the combination of Y181C and E138K may lead to a less fit virus. In our study, amino acid substitutions at position 179 were frequently observed in viruses containing Y181C at baseline. This pathway has also been observed [38,40].
The complex formed between RPV and wild-type reverse transcriptase is stable and comparable to the complex between RPV and K103N mutant reverse transcriptase. In the presence of RPV and mutant reverse transcriptase containing Y181C, there is a loss of aromatic interactions, resulting in a displacement of RPV further into the NNRTI pocket . We believe that differences in binding interactions that exist between RPV or ETR and the Y181C reverse transcriptase mutant may be the basis for potential antagonism.
Although the prevalence of at least three ETR RAMs among viral isolates from patients experiencing treatment failure under EFV and NVP therapy was low, ranging from 4.6 to 10%, the prevalence of single ETR RAMs was high (17.4 to 35.9%) [11,26,42]. These studies concluded that there is a low prevalence of ETR resistance at baseline, and that patients with prior failure to EFV and NVP could potentially benefit from ETR therapy. However, these data were obtained in developed countries in which full access to potent antiretroviral drugs is the norm. Studies in resource-limited settings have shown a high prevalence of many NNRTI resistance mutations associated with ETR resistance among patients failing EFV or NVP, questioning the potential effectiveness of ETR and RPV in such settings [43–45]. There is concern that patients, who fail RPV, because of the E138K mutation, will be unlikely to derive future benefit from ETR.
In summary, we have shown that each of ETR and RPV are likely to select for E138K as a major resistance mutation in tissue culture if no or very few other resistance mutations are present. In contrast, viruses containing an array of other common NNRTI mutations associated with resistance to EFV and NVP, such as Y181C, may be less likely to select E138K following exposure to ETR and RPV, and may develop other substitutions instead. In part, this may be due to diminished viral replicative capacity. These tissue culture observations are consistent with the clinical experience and help to explain why the resistance profile of ETR as a second-line drug is different from what it might have been if this drug were to be more commonly employed in first-line therapy. However, clinical studies with ETR and RPV in NNRTI-naive and NNRTI-experienced patients will be necessary to validate the present in-vitro findings.
E.L.A., M.A.W., and C.L.T. conceived the hypothesis and designed this study. E.L.A. and X.H.T. engineered the clones. E.L.A. and M.O. performed all culture work. E.L.A., D.M., and I.R.I. performed sequencing and analyzed the data. E.L.A., M.A.W., and C.L.T. wrote the paper. B.B.G. critiqued the paper.
This research was supported by grants from the Canadian Institutes of Health Research and the Réseau SIDA-MI of the Fonds de la Recherche en Sante du Quebec. E.L.A. is the recipient of a Departmental Scholarship from the Département de Microbiologie et d’Immunologie, Université de Montréal. C.T. is the Pfizer/Université de Montréal Chair on HIV Translational Research.
Conflicts of interest
There are no conflicts of interest.
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This article has been cited 3 time(s).
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antiviral activity; cross-resistance; etravirine; replication capacity; rilpivirine; second generation nonnucleoside reverse transcriptase inhibitor
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