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Antimicrobial Agents and Chemotherapy, September 2000, p. 2475-2484, Vol. 44, No. 9
DuPont Pharmaceuticals Company, Experimental
Station, Wilmington, Delaware 19880-0336
Received 11 February 2000/Returned for modification 13 April
2000/Accepted 22 June 2000
Efavirenz is a potent and selective nonnucleoside inhibitor of
human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT).
Nucleotide sequence analyses of the protease and RT genes (coding
region for amino acids 1 to 229) of multiple cloned HIV-1 genomes from
virus found in the plasma of patients in phase II clinical studies of
efavirenz combination therapy were undertaken in order to identify the
spectrum of mutations in plasma-borne HIV-1 associated with virological
treatment failure. A K103N substitution was the HIV-1 RT gene mutation
most frequently observed among plasma samples from patients for whom
combination therapy including efavirenz failed, occurring in at least
90% of cases of efavirenz-indinavir or efavirenz-zidovudine
(ZDV)-lamivudine (3TC) treatment failure. V108I and P225H mutations
were observed frequently, predominantly in viral genomes that also
contained other nonnucleoside RT inhibitor (NNRTI) resistance
mutations. L100I, K101E, K101Q, Y188H, Y188L, G190S, G190A, and G190E
mutations were also observed. V106A, Y181C, and Y188C mutations, which
have been associated with high levels of resistance to other NNRTIs,
were rare in the patient samples in this study, both before and after
exposure to efavirenz. The spectrum of mutations observed in cases of
virological treatment failure was similar for patients initially dosed
with efavirenz at 200, 400, or 600 mg once a day and for patients
treated with efavirenz in combination with indinavir, stavudine, or
ZDV-3TC. The proportion of patients carrying NNRTI resistance
mutations, usually K103N, increased dramatically at the time of initial
viral load rebound in cases of treatment failure after exposure to
efavirenz. Viruses with multiple, linked NNRTI mutations, especially
K103N-V108I and K103N-P225H double mutants, accumulated more slowly
following the emergence of K103N mutant viruses.
The reverse transcriptase (RT) of
human immunodeficiency virus type 1 (HIV-1) is critical to the life
cycle of HIV and is without a homologue in eukaryotic organisms. As
such, it is an attractive target for selective antiviral therapy. Among
inhibitors of RT, a large class of chemically diverse, generally
HIV-1-specific, nonnucleoside reverse transcriptase inhibitors (NNRTIs)
has been identified. These inhibitors generally act by binding to a
site on the RT that is distinct from the polymerase catalytic site. While NNRTIs can be potent inhibitors of HIV-1 replication, with favorable safety and pharmacokinetic parameters, rapid emergence of
resistant viruses both in vitro (20, 24) and in vivo
(11, 14, 15, 25, 30), often as the result of single
nucleotide changes, has limited the therapeutic utility of these
compounds as monotherapy. However, recent clinical trials of the use of NNRTIs in combination with other antiretroviral agents have
demonstrated an added benefit from inclusion of an NNRTI in such
combination regimens (5, 6, 12, 21, 29, 30; S. Green, M. F. Para, P. W. Daly, W. W. Freimuth, L. D. Getchel, C. A. Greenwald, and L. K. Wathen, 12th World
AIDS Conference, Geneva, Switzerland, abstr. 12219, 1998). A variety of
mutations in the HIV-1 RT gene associated with resistance to NNRTIs
have been identified (2, 23, 27). In models of the
three-dimensional structure of HIV-1 RT (28), these
mutations cluster around the NNRTI binding site.
Efavirenz is a potent inhibitor of HIV-1 replication. In clinical
studies, efavirenz used once daily in combination with nucleoside analogs or protease inhibitors has shown potent antiviral activity and
significant clinical efficacy (29, 30). In cell culture, efavirenz retains significant activity against a variety of mutant strains of HIV-1 with single amino acid substitutions in the RT gene
which have been associated with resistance to other NNRTIs (4,
33; L. Bacheler, unpublished data). Cell culture selection experiments (32) demonstrated that passage of the RF strain of HIV-1 in MT-2 cell or peripheral blood mononuclear cell culture led
to the selection of mutations at amino acid positions 100, 108, 179, and 181 of the HIV-1 RT gene. Young et al. (33) reported the
selection in cell culture of an L100I-K103N double mutant that was
highly resistant to efavirenz. In both cases, high-level resistance to
efavirenz ( In the studies reported here, we determined the nucleotide sequence of
the protease and RT genes of plasma-borne HIV-1 from patients in three
phase II dose-ranging studies of efavirenz combination therapy. These
studies allowed us to assess the potential for selection of
drug-resistant virus variants in vivo during efavirenz combination
therapy and to identify the nature and time course of the emergence of
such mutations relative to rebounds in plasma virus load, which might
be indicative of loss of therapeutic efficacy.
Selection of plasma samples and clonal sequencing approach.
Plasma samples collected at baseline and at intervals before and after
a significant rebound in plasma virus load from efavirenz-exposed patients in clinical studies DMP 266-003, -004, and -005 (Table 1) were selected for sequencing. As per
clinical study protocol, cases where a patient's plasma HIV RNA level,
as determined by the Roche Amplicor HIV-1 Monitor assay, rose above
1,000 copies/ml of plasma on two successive occasions after reaching a
nadir of 400 or fewer copies/ml or, if the plasma HIV RNA level never
dropped below 400 copies/ml, where the level rose
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Human Immunodeficiency Virus Type 1 Mutations
Selected in Patients Failing Efavirenz Combination Therapy
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
100-fold increase in 90% inhibitory concentration)
appeared to be associated with multiple mutations in the RT gene of
HIV-1.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
0.75 log units
relative to the lowest RNA copy number previously measured on two
successive visits were defined as virological treatment failures. In
addition, baseline plasma samples from some patients who experienced a
sustained suppression in viral load were selected for sequencing.
TABLE 1.
Phase II clinical studies of efavirenz combination
therapy in which virological treatment failures
were characterizeda
Extraction of RNA from plasma samples. RNA was extracted from 200-µl plasma samples using the RNAgents Total RNA Isolation System kit (Promega Corp.). RNA precipitated by isopropanol was washed twice with 80% ethanol and dried for 5 min under vacuum at room temperature.
RT-PCR amplification. The RNA pellet was resuspended in 120 µl of RT solution (0.5 U of Superscript II RT per ml, 1× Superscript II buffer [Gibco/BRL, Gaithersburg, Md.], 10 mM dithiothreitol [Gibco/BRL], 0.75 mM [each] dATP, dCTP, dGTP, and dTTP [Perkin-Elmer], 1 U of human placental RNase inhibitor [Boehringer Mannheim] per µl, and 1 µM primer P8 [5' TAAATCTGACTTGCCCAATTCAATTTT 3', corresponding to nucleotides 3335 to 3361 of HIV strain HXB2; Midland Certified Reagent Co.]). The mixture was allowed to dissolve for 5 min at room temperature and then incubated at 50°C (or at 42°C in some experiments) for 1 h.
The cDNA products of the reverse transcription reaction were used as the template in 12 nested PCRs to amplify a 1.1-kb region of HIV-1 containing both the protease gene and the 5' portion of the RT gene. In the first PCR, 10 µl of the RT reaction mixture was added to 40 µl of PCR I mix consisting of 1 µl of TaqStart antibody (Clonetech), 1 µl of TaqPlus Long DNA polymerase (Stratagene) (these two reagents were mixed for 5 min before being added to the remaining reagents), 4 µl of 10× TaqPlus Long low-salt buffer (Stratagene), 1 µl of 12.4 mM primer P7 (5' AGACCAGAGCCAACAGCCCCAC 3', corresponding to nucleotides 2143 to 2164 of HIV strain HXB2; Midland Certified Reagent Co.), and 33 µl of water. The cycling conditions were an initial incubation at 95°C for 5 min, followed by 35 cycles of 94°C for 15 s, 56°C for 1 min, and 72°C for 2 min, with a final 6 min at 72°C. Second-round PCRs were performed using primer P9-amp1 (5' CUACUACUACUAAGCCGATAGACAAGGAACTGTA 3', corresponding to nucleotides 2219 to 2240 of HIV strain HXB2; Midland Certified Reagent Co.) and P10-amp1 (5' CAUCAUCAUCAUCAGGATGGAGTTCATAACCCAT 3', corresponding to nucleotides 3237 to 3258 of HIV strain HXB2; Midland Certified Reagent Co.). Fifty microliters of PCR II mix (1 µl of TaqStart antibody plus 1 µl of TaqPlus Long DNA polymerase (mixed as described above), 5 µl of 10× TaqPlus Long low-salt buffer, 1 µl each of 10 mM dATP, 10 mM dCTP, 10 mM dGTP, and 10 mM dTTP, 1 µl of 100 mM P9-amp1 and 1 µl of 100 mM P10-amp1) was added to the initial 50-µl PCR mixture and amplified for 35 cycles of 94°C for 15 s, 52°C for 1 min, and 72°C for 2 min, with a final 6 min at 72°C. Five microliters of each PCR mixture were run on a 1% agarose gel to confirm the presence of a 1.1-kb product and the absence of products of similar sizes in extracted and amplified samples of seronegative human plasma. Multiple (usually 12) nested PCRs were performed with the cDNA from each plasma sample, with the goal of obtaining one HIV-1 sequence from each of eight independently amplified viral genomes.Purification of PCR products. PCR amplification products were purified using solid-phase, reversible immobilization on carboxy-coated magnetic particles (PerSeptiveBiosystems, Inc., Framingham, Mass.) as described by DeAngelis et al. (10). Briefly, 45 µl of each PCR mixture was mixed with 50 µl of binding buffer (2.5 M NaCl-20% polyethylene glycol) and 10 µl of SPRI beads at a concentration of 10 mg/ml (previously washed 3 times with 0.5 M EDTA [pH 8.0]). Samples were incubated at room temperature for 10 min, washed twice with 150 µl of 70% ethanol, and air dried for 2 min. DNA was eluted from the beads with 20 µl of elution buffer (10 mM Tris-acetate [pH 7.8]). The particles were magnetically separated, and the supernatants were removed and retained. For selected samples, PCR products were purified following electrophoresis on 1% agarose gels. The desired band (approximately 1,200 bp) was excised from the gel with the tip of a sterile Pasteur pipette. The resulting 5- to 10-µl agarose gel plug containing the PCR product was expelled into 7.5 µl of 10 mM Tris-acetate (pH 7.8).
Cloning of PCR products.
Purified PCR products were inserted
into the cloning vector pAMP-1 (CloneAmp System; Gibco/BRL), which
utilizes uracil DNA glycosylase to facilitate directional cloning of
PCR products. Annealed DNA samples were subsequently transformed into
Escherichia coli strain DH5
(DH5 maximum efficiency;
Gibco/BRL) using the procedure of Hanahan (13).
Preparation of plasmid DNA. Purification of plasmid DNA from individual E. coli clones was performed on a BioRobot 9600 (Qiagen Inc., Chatsworth, Calif.) using Qiawell Ultra plasmid kits based on the optimized alkaline lysis method of Birnboim (3). Following cell growth in Luria broth containing 150 µg of ampicillin per ml, cells were pelleted and loaded onto the BioRobot 9600 for processing using Qiasoft version 2.0 software.
Nucleic acid sequencing. The cloned DNAs were sequenced from double-stranded plasmids using six primers that spanned the cloned protease and RT genes (all primers were from Midland Certified Reagent Co.). Three primers (pSP8 [5' GGTGACACTATAGAAGAGCT 3', complementary to the cloning vector], p517F [5' GGATGGCCCAAAAGTTA 3', corresponding to nucleotides 2597 to 2613 of the HXB2 strain of HIV-1], and p743F [5' CAATTAGGAATACCACATCC 3', corresponding to nucleotides 2820 to 2839 of the HXB2 strain of HIV-1]) were used to sequence in the forward direction, and three primers (pT7 [5' TAATACGACTCACTATAGGG 3', complementary to the cloning vector], p461R [5' TGGTACAGTTTCAATAGGAC 3', corresponding to nucleotides 2557 to 2576 of the HXB2 strain of HIV-1], and p2A [5' CCACATCCAGTACTGTTACTG 3', corresponding to nucleotides 2863 to 2883 of the HXB2 strain of HIV-1]) were used to sequence in the reverse direction.
Thermal cycle sequencing reactions were carried out using 0.5-µg samples of the DNA templates and 20 ng of each primer. Polymerase TaqFS and either Big Dye or d-rhodamine dye terminators were used for sequencing; purified sequencing reaction were run on a Perkin-Elmer/Applied Biosystems 377 DNA sequencer. Consensus sequences for each clone were assembled using Sequencher software (Gene Codes Corporation).Database. Consensus DNA sequences were exported from Sequencher as text files. These files were translated into protein sequences for the HIV protease and RT genes. The region sequenced covers the coding region for amino acids 1 to 99 of protease and amino acids 1 to 229 of RT. The protein and nucleotide sequence information was imported into an Oracle database that linked the sequence information from each clone to the corresponding patient, clinical trial, and time point at which the sample was isolated. Sequences were analyzed following alignment of the protease and RT sequences from individual clones; comparisons were made between these sequences and the clade B consensus sequence (22). Sequences were analyzed by database query for the presence of mutations previously reported to be associated with the development of resistance to any NNRTI (2, 23, 27) and were visually examined for the emergence of novel mutations after exposure to efavirenz.
BLAST searches. Each nucleotide sequence was compared (by a BLASTN [version 1.4.7] [1] search) to the entire set of HIV sequences derived from patient samples, as well as to sequences of laboratory strains of HIV-1, including those handled in the laboratory in which plasma samples were extracted and amplified. Individual sequences were automatically flagged for further scrutiny if (i) among 10 or more sequences from the same patient in the database, 5 or fewer were among the top 10 matches to the test sequence; (ii) a sequence from a wild-type laboratory strain was among the top 10 matches to the test sequence, and the wild-type strain matched the test sequence more closely than it did other sequences from the same patient; or (iii) the test sequence was divided into two high-scoring segment pairs (an n = 2 in the BLASTN search results), indicative of a frameshift or deletion in one of the two sequences. Flagged sequences, as well as a random sampling of nonflagged sequences, were reviewed. Sequences with insertions, deletions, or frameshifts were allowed if the amino acid translation of the remainder of the sequence showed high homology to other sequences from the same patient. Sequences which showed closer homology to sequences from a different patient or to sequences of laboratory strains of HIV-1, suggestive of cross-contamination, were tagged in the Oracle database and excluded from the set of sequences queried to calculate the data reported here.
Nucleotide sequence accession numbers. Nucleotide sequences determined in this study have been deposited in GenBank under accession numbers AY000001 to AY003708.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The emergence of genotypic resistance to efavirenz was studied in samples collected during three clinical studies of efavirenz combination therapy summarized in Table 1. These phase II studies tested efavirenz in combination with the protease inhibitor indinavir or the nucleoside analog RT inhibitors (NRTIs) zidovudine (ZDV, also known as AZT) and lamivudine (3TC). Both NRTI-naïve and -experienced patients were enrolled in these studies, but all patients were required to be NNRTI and protease inhibitor (PI) therapy naïve. Two cohorts (21 patients) of study DMP 266-003 began with a 2-week efavirenz monotherapy lead-in period before combination therapy was started.
As an example of the clonal sequencing approach, results from a single
patient are shown. The viral load response of patient 22, who received
efavirenz monotherapy at 200 mg once a day (q.d.) for 14 days before
the initiation of efavirenz-indinavir combination therapy, is shown in
Fig. 1. Table
2 shows variations from the clade B amino
acid consensus sequence observed at selected positions in the RT gene
of virus isolated from the plasma of patient 22 and cloned. At
baseline, this patient's mixed plasma virus population included
variations at amino acids 98 and 211 of RT and at amino acid 77 of
protease (protease gene data not shown). After only 14 days of
efavirenz monotherapy, K103N mutations in RT were observed in 2 out of
10 cloned viral genomes. Despite the emergence of a K103N mutant virus,
initiation of efavirenz-indinavir combination therapy led to a further
reduction in viral load (to fewer than 400 copies/ml of plasma), which
reduced level was maintained until day 349. At this time, HIV-1 RNA in
plasma began to increase, eventually returning to close to baseline
levels. At day 349, sequencing revealed a virus population carrying
K103N mutations in RT and V82A mutations in protease in all 4 clones
sequenced. At day 424, as the viral load continued to rebound, K101E
and P225H mutations in RT, in combination with K103N or K103E
mutations, were observed, while L63P and M46L mutations were observed
in combination with V82A mutations in protease. By day 507, approximately 158 days after the beginning of viral load rebound,
K103N-P225H double RT mutants were more prevalent (observed in 4 of 8 clones). In protease, additional mutations at positions 54 and 71 of
the protease gene were detected.
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, time point
at which plasma virus genotype was determined. The timing of efavirenz
monotherapy and efavirenz plus indinavir combination therapy is
indicated by the bars. EFV, efavirenz; IDV, indinavir; q8h, every
8 h.
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In Table 3, a summary of the frequencies
of mutations previously reported to be associated with resistance to
one or more NNRTIs among plasma samples collected at baseline (prior to
exposure to any study medications) and during the study for patients
experiencing virological treatment failure in study DMP 266-003 is
provided. In this study, patients were treated with the PI indinavir
plus efavirenz or efavirenz placebo. Some patients, including patient 22 (described above), received efavirenz monotherapy for 2 weeks before
efavirenz-indinavir combination therapy was initiated. Other patients
initially randomized to indinavir monotherapy were later allowed to
switch to efavirenz-D4T combination therapy. Some of these patients
failed this second drug regimen, and a genotypic characterization of
virus from such "switched" patients is included in Table 3.
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As shown in Table 3, mutations previously described as associated with resistance to one or more NNRTIs were infrequently detected in plasma virus from the NNRTI-naïve patients enrolling in this study. The mutations that were detected were frequently seen in a single clone per patient sample. Similarly, mutations associated with resistance to one or more NNRTIs were infrequent among samples collected during the study from patients receiving efavirenz placebo (i.e., indinavir monotherapy) who experienced a rebound in viral load. In contrast, a variety of mutations previously described as associated with NNRTI resistance was detected in plasma samples collected during the study from patients who experienced virological treatment failure of an efavirenz-containing drug regimen. K103N was the most frequently observed NNRTI resistance mutation in samples from efavirenz-exposed patients who experienced a significant rebound in viral load, detected in more than 90% of cases of efavirenz treatment failure. This amino acid substitution can be achieved by a single base pair change or point mutation (AAG or AAA to AAC or AAT). V108I and P225H mutations were observed in 37.5 and 40.6%, respectively, of efavirenz-exposed patients who experienced treatment failure and at similar frequencies (21.4 and 50%, respectively) among patients initially randomized to efavirenz placebo who later switched to an efavirenz-containing regimen and for whom the second regimen failed. Additional mutations observed in multiple clones from a significant number of patients for whom combination therapy with efavirenz failed included L100I, K101E, K101Q, Y188H, Y188L, and G190S. Mutations at Y181C, Y188C, or V106A, which are associated with high levels of resistance to nevirapine and/or delavirdine, were observed only rarely, often in a single cloned viral genome from a given patient.
Mutations associated with resistance to one or more PIs were more frequently detected at baseline than those associated with resistance to NNRTIs, although most of the mutations detected are considered secondary resistance mutations (16) and are recognized as naturally occurring polymorphisms in the highly variable HIV-1 protease gene (17). Primary PI resistance mutations (D30N, M46I, M46L, M46V, G48V, I50V, V82A, V82F, and V82T, as defined by Hirsch et al. [16]) were only infrequently detected, usually in a single clone per patient sample. Viral sequences collected from patients who experienced treatment failure with either indinavir monotherapy or efavirenz-indinavir combination therapy had an increased prevalence of a variety of mutations associated with resistance to PIs, including mutations at amino acid positions 46 and 82, which have been associated with phenotypic resistance to indinavir (8). Secondary PI mutations detected at increased frequency in viral genomes from patients who had failed indinavir therapy included L10F, L10I, L10R, L10V, L24I, A71V, G73S, and L90M. The spectrum of PI resistance mutations observed did not appear to vary between patients for whom indinavir monotherapy failed and those for whom efavirenz-indinavir combination therapy failed.
Since nucleotide sequence information was determined for cloned HIV-1
genomes, the linkage of multiple mutations in the same DNA clone could
be identified. These linked mutations represent sequence variations
present in the same viral RNA genome prior to RT-PCR amplification.
Table 4 compares the prevalence of
specific, single NNRTI mutations and linked, multiple NNRTI mutations
in 161 baseline samples collected from patients entering studies DMP
266-003, -004, and -005 to the prevalence of the same mutations in
samples collected from 104 of these patients after a significant rebound in viral load. As in study DMP 266-003, the prevalence of NNRTI
mutations was low at study entry. Among cases of efavirenz treatment
failure, the K103N mutation was identified in cloned HIV-1 genomes both
as a single NNRTI mutation and in combination with other NNRTI
mutations. Many patients had viruses with K103N both as a single
mutation (88.5% of patients) and in combination with other NNRTI
resistance mutations (64.4% of patients), either in sequential samples
or within a single plasma sample. In contrast, L100I, K101Q, V108I,
Y188H, and P225H mutations were seen largely or exclusively in
combination with other NNRTI resistance mutations, not as single NNRTI
resistance mutations. While a variety of viral genotypes combining one
or more mutations previously associated with NNRTI resistance were
identified, combinations containing K103N were by far the most
prevalent. The most frequently observed double NNRTI mutants include
K103N-V108I and K103N-P225H, each observed in approximately one-third
of efavirenz treatment failures. Other double mutants observed in more
than 10% of efavirenz treatment failures include L100I-K103N and
K101Q-K103N. A triple mutant virus carrying K103N, V108I and P225H
mutations was observed in 9.6% of patients for whom efavirenz
combination therapy failed. K101E mutations were observed in 13.8% of
efavirenz treatment failure patients overall, either as a single NNRTI
resistance mutation (4.8% of patients) or linked to a variety of other
NNRTI resistance mutations (not just K103N). Viruses with Y188L
mutations, often linked to V106I mutations, were observed in a small
percentage of patients. Y188L mutated viruses appear to represent an
alternate, infrequently utilized path to efavirenz resistance; K103N
mutant viruses were infrequently observed in patients who developed
Y188L mutant viruses.
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In the three phase II clinical trials studied, patients were dosed with
three different doses of efavirenz (200, 400, or 600 mg q.d.). Some
patients initially dosed at 200 mg in study DMP 266-003 had the dose of
efavirenz increased to 600 mg q.d. during the study. The pattern of
NNRTI mutations observed in cases of efavirenz treatment failure was
examined for the three doses of efavirenz tested (Fig.
2A). K103N was the most frequent NNRTI resistance mutation observed in the plasma virus of patients who experienced virological treatment failure and who were initially dosed
at 200, 400, or 600 mg q.d. of efavirenz. V108I and P225H mutations,
most often in combination with K103N, were frequent at each dose of
efavirenz studied. K101E and K101Q mutations were observed for more
than 10% of cases of efavirenz treatment failure at each efavirenz
dose. The types and relative frequency of NNRTI mutations selected in
patients for whom efavirenz combination therapy failed was also
examined as a function of the coadministered study medications. Data
were available for patients treated with efavirenz in combination with
indinavir, with D4T or 3TC, or with ZDV-3TC in both NRTI-experienced
and -naïve patients. Figure 2B illustrates that a similar
pattern of selected NNRTI resistance mutations was observed for each
drug combination.
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Since multiple plasma samples from patients for whom efavirenz
combination therapy failed collected before and at various times during
the rebound in viral load were analyzed, it was possible to examine the
time of appearance of viruses with single versus multiple mutations.
Figure 3A shows the time of appearance of virus with one, two, or three linked NNRTI mutations relative to the
time of initial rebound in viral load used to define virological treatment failure for patients in study DMP 266-004. The proportion of
treatment failure patients whose plasma virus carried at least one
NNRTI resistance mutation increased dramatically around the time of
initial rebound in viral load, from 20% of patient samples tested 57 or more days prior to the beginning of viral load rebound to
approximately 75% of patient samples tested at the time of initial
viral load rebound (failure day). By 100 days after the beginning of
viral load rebound, more than 50% of patients had viruses with two
NNRTI mutations in the same viral genome, and 15% of patients had
viruses with three linked NNRTI mutations. A similar pattern of early
emergence of single and later emergence of double or triple NNRTI
resistance mutations was also observed in cases of efavirenz treatment
failure in studies DMP 266-003 and -005, although the number of
patients who experienced treatment failure in study DMP 266-005 whose
plasma virus was characterized was small (10 patients).
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In study DMP 266-003, the proportion of patients that had viruses with NNRTI resistance mutations before the viral load rebounded and who experienced treatment failure was larger than that in study DMP 266-004 (data not shown). This early appearance of NNRTI resistance mutations was observed mainly among patients in the two efavirenz monotherapy cohorts. Sequence data from plasma samples collected after 2 weeks of efavirenz monotherapy at 200 mg q.d. were available for 16 patients. At the end of this short monotherapy period, viruses with one or more NNRTI mutations were detected, although often as a minority of the clones sequenced, in 11 of 16 patients. For many of these monotherapy patients, addition of indinavir at the end of 2 weeks led to further suppression of plasma virus load. Overall, however, the proportion of patients who began with short-term efavirenz monotherapy and eventually experienced virological treatment failure was higher than that of patients who always received efavirenz in combination with other antiretroviral drugs.
Figure 3B shows the accumulation of specific NNRTI resistance mutations as a function of time before or after the beginning of viral load rebound in cases of efavirenz treatment failure in study DMP 266-004. The K103N mutation was detected in the majority of patients at or shortly after the initial rebound in viral load. V108I and P225H mutations, arising largely in combination with K103N mutations, accumulated more gradually with increasing time after the beginning of viral load rebound. As demonstrated in vitro (32, 33; L. T. Bacheler, unpublished data), these combinations of multiple NNRTI mutations are likely to confer higher levels of resistance to efavirenz beyond that associated with the initial K103N mutation.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the studies reported here, we adopted a clonal sequencing approach for the detection of mutations in plasma-borne HIV-1 that might be associated with resistance to efavirenz. Multiple independently amplified and cloned viral genomes were sequenced from each plasma sample. This methodology offers some unique advantages for the characterization of resistance to a new antiretroviral agent. Sequencing multiple clones facilitates the detection of minority viral species, including those associated with emerging viral resistance (7, 19). In many cases, we first identified a K103N mutation in one or two clones out of eight derived from a single plasma sample and then identified a K103N mutation in all clones derived from a second, later plasma sample. Sequencing a single clone from each independent PCR amplification avoids founder effects that could distort the representation of specific genotypes in the collection of sequences derived from a plasma sample. The linkage of specific mutations in a single viral genome is readily detected by this approach. Certain mutations associated with resistance to efavirenz were largely or exclusively detected linked to other mutations. The L100I mutation, while conferring resistance to efavirenz in constructed viruses as a single mutation (34; L. T. Bacheler, unpublished data), was never observed as the only NNRTI resistance mutation in plasma virus from patients for whom efavirenz therapy failed; rather, this mutation was found linked to a K103N mutation. The frequently observed V108I and P225H mutations, which do not confer detectable resistance to efavirenz in vitro as single mutations (L. T. Bacheler, unpublished data) were also found predominantly in viral genomes also carrying K103N mutations. In some plasma samples, a diverse collection of NNRTI resistance mutations was detected. Only through a clonal sequencing approach could associations between specific mutations be discerned. There are, however, several disadvantages to the clonal sequencing method. First, it is very labor intensive. Second, since single cloned viral genomes are sequenced, this method may identify mutations introduced during PCR amplification, which do not reflect the initial plasma virus population. The viral origin of rare mutations, such as Y181C in patients for whom efavirenz therapy failed or a K103N mutation in a plasma sample collected before exposure to efavirenz, must remain in some doubt because of this technical drawback. For this reason we have chosen to focus on mutations detected either in virus from plasma of at least two patients or in multiple clones in virus from plasma of a single patient. Even rare mutations were, however, embedded in viral sequence with the highest homology by BLASTN search to other sequences derived from the same patient and are therefore unlikely to represent PCR contamination from other patient samples or laboratory strains.
Many of the Phase II patients in whom efavirenz resistance has been characterized were treated with regimens which would be considered suboptimal by present standards. Two weeks of efavirenz monotherapy prior to the initiation of combination therapy, suboptimal doses of efavirenz (e.g., 200 or 400 mg q.d.), and addition of efavirenz as a single new antiretroviral agent to a submaximally suppressive regimen (e.g., ZDV-3TC-experienced patients with active viral replication in study DMP 266-004) each led to virological treatment failure and the emergence of efavirenz-resistant virus in some patients. Nevertheless, the pattern of NNRTI resistance mutations selected in these patients appears to be similar to that observed in patients treated with the recommended clinical dose of 600 mg of efavirenz q.d. In addition, the pattern of resistance mutations associated with efavirenz treatment failure was similar in regimens combining efavirenz with a PI (indinavir) or dual nucleoside analogs (ZDV-3TC or D4T-3TC). In contrast, different patterns of resistance mutations have been reported for patients for whom nevirapine combination therapy failed, depending upon the coadministered nucleoside analogs. While nevirapine monotherapy rapidly selects for Y181C mutations, mutations at RT positions 103, 106, 188, and 190 were observed in patients failing nevirapine plus ZDV combination therapy, while fewer mutations were found at position 181 (6, 15). This result may be explained in part by the observation that the Y181C mutation significantly enhances susceptibility to AZT in molecular constructs carrying this mutation in an AZT resistant background (4, 18). Efavirenz is fully active in vitro against virus constructs with a Y181C mutation (33; L. T. Bacheler, unpublished data). Thus, Y181C mutations are unlikely to be selected by efavirenz combination therapy, with or without coadministered ZDV.
Like other NNRTIs studied in vivo, efavirenz appears to rapidly select for resistant viruses when used in suboptimally suppressive regimens. More than half of the patients treated with efavirenz monotherapy (200 mg q.d.) for 14 days had developed resistance mutations at the end of this short monotherapy period; the mutations were detected by our clonal sequencing approach, albeit as minority components of the virus population. Some patients in study DMP 266-004, who added efavirenz at 200 or 400 mg q.d. to a preexisting dual nucleoside regimen which was not maximally suppressing HIV replication also experienced early viral load rebound associated with the emergence of NNRTI resistance mutations. Both of these observations highlight the importance of avoiding the use of actual or functional efavirenz monotherapy, such as when efavirenz is added as a single agent to a failing drug regimen, in order to avoid the selection of resistant viruses.
While the point mutation K103N was most often the first NNRTI resistance mutation observed in patients for whom efavirenz combination therapy failed, the majority of patients studied went on to develop viruses carrying multiple mutations. Indeed, a number of the most prevalent mutations observed (V108I, P225H, K101E or K101Q, and L100I) were almost exclusively observed as double mutants in combination with K103N. In cases where the effect on drug susceptibility of such double mutants has been tested, the double mutants are associated with higher levels of resistance to efavirenz. (L. T. Bacheler, unpublished data). These data suggest that efavirenz continues to exert selective pressure on K103N mutant strains, resulting in the eventual selection of double- and even triple-mutant viruses which have higher levels of drug resistance than that of a K103N single-mutant virus.
The spectrum of resistance mutations observed in vivo in patients for whom efavirenz combination therapy failed, as well as their relative frequency, was substantially different from that observed in cell culture selection experiments. The L100I-K103N double mutant selected in cell culture (33) was relatively infrequently observed in vivo, occurring in 10.6% of virological failures. In the selection experiments described by Winslow et al. (32) viruses with mutations at L100, V108, V179, and Y181 were described. In vivo, the L100I mutation was seen only in combination with K103N, and V108I mutations were observed largely in combination with K103N. V179D and Y181C mutations were extremely rare in plasma virus from patients exposed to efavirenz. The results of our characterization of the genotypic correlates of resistance to efavirenz in vivo emphasize that cell culture selection experiments are often not predictive of the types or frequencies of resistance mutations selected in patients receiving antiretroviral therapy. Similar discordancies between resistance mutations selected in vivo and in vitro have been described for delavirdine (11) and the experimental NNRTI HBY 097 (26).
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ACKNOWLEDGMENTS |
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We thank Jon Condra and Emilio Emini of Merck Research Laboratories for discussions and initial sequencing.
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FOOTNOTES |
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* Corresponding author. Mailing address: DuPont Pharmaceuticals Company, E336/36B Experimental Station, Wilmington, DE 19880-0336. Phone: (302) 695-4278. Fax: (302) 695-9466. E-mail: lee.bacheler{at}dupontpharma.com.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Wirden, M., Simon, A., Schneider, L., Tubiana, R., Paris, L., Marcelin, A. G., Delaugerre, C., Legrand, M., Herson, S., Peytavin, G., Katlama, C., Calvez, V.
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