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Antimicrobial Agents and Chemotherapy, March 2009, p. 1116-1123, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01055-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Virologic Failure in First-Line Human Immunodeficiency Virus Therapy with a CCR5 Entry Inhibitor, Aplaviroc, plus a Fixed-Dose Combination of Lamivudine-Zidovudine: Nucleoside Reverse Transcriptase Inhibitor Resistance Regardless of Envelope Tropism

James F. Demarest,^* Heather Amrine-Madsen, David M. Irlbeck, Kathryn M. Kitrinos, on behalf of the CCR102881 Clinical Study Team

GlaxoSmithKline, Research Triangle Park, North Carolina 27709

Received 5 August 2008/ Returned for modification 26 September 2008/ Accepted 29 November 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The CCR102881 (ASCENT) study evaluated the antiviral activity of the novel CCR5 entry inhibitor aplaviroc plus a fixed-dose combination of lamivudine-zidovudine (Combivir) in drug-naïve human immunodeficiency virus type 1-infected subjects with only CCR5-tropic virus detected in plasma. Although the trial was stopped prematurely due to idiosyncratic hepatotoxicity, eight subjects met protocol-defined virologic failure criteria. Clonal analyses of the viral envelope tropism, aplaviroc susceptibility, and env sequencing were performed on plasma at baseline and at the time of virologic failure. Molecular evolutionary analyses were also performed. The majority of the subjects with virologic failure (six of eight) acquired the lamivudine resistance-associated mutation M184V, and none had evidence of reduced susceptibility to aplaviroc at the time of virologic failure, even at the clonal level. Six subjects with virologic failure maintained CCR5 tropism, while two exhibited a change in population tropism readout to dual/mixed-tropic with R5X4-tropic clones detected prior to therapy. Two evolutionary patterns were observed: five subjects had no evidence of population turnover, while three subjects had multiple lines of evidence for env population turnover. The acquisition of the M184V mutation is the primary characteristic of virologic failure in first-line therapy with aplaviroc plus lamivudine-zidovudine, regardless of the envelope tropism.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Despite the clinical benefits of highly active antiretroviral therapy (HAART), drug side effects and the emergence of multidrug-resistant viral strains demonstrate the need for alternative approaches to human immunodeficiency virus (HIV) therapy (11, 27). The human chemokine receptors CCR5 and CXCR4 may also function as coreceptors for HIV-1 entry into CD4⁺ cells (2). As such, coreceptor inhibitors are being developed as novel approaches for antiretroviral therapy (ART). For example, the CCR5 antagonist maraviroc (Pfizer United Kingdom, Sandwich, Kent, England) was recently approved for use in HIV-positive treatment-experienced patients.

At the individual virus level, coreceptor tropism refers to the ability of an HIV-1 envelope to utilize CCR5 (R5-tropic), CXCR4 (X4-tropic), or both (R5X4-tropic) for entry into CD4⁺ cells. A virus population, such as that found in patient plasma, may comprise R5-, R5X4-, and/or X4-tropic envelopes. Such samples are referred to as dual/mixed (DM) or DM-tropic and require clonal analyses to characterize the relative frequencies of viruses with a particular tropism in the viral population. The data available to date on tropism prevalence shows that the majority of HIV-1⁺ patients test as R5-tropic (>80% of ART-naïve and 50 to 70% of ART-experienced patients [3, 4, 7, 20, 21, 33, 35]). The remaining patients test as DM-tropic with only a small percentage (<5%) as X4-tropic, almost exclusively in the ART-experienced population (4, 7, 20, 21, 33, 35). Thus, the vast majority of HIV⁺ patients harbor CCR5-using viruses and may benefit from HAART containing CCR5 entry inhibitors (EIs).

The CCR5 EI GW873140, or aplaviroc (APL), showed in vitro antiviral activity against R5-tropic HIV-1 at subnanomolar concentrations (8, 9, 19). A 10-day study of APL monotherapy in HIV-1-positive subjects demonstrated a 1.66 log₁₀ copies/milliliter (c/ml) mean decrease at the nadir of the viral load from baseline for the dosage arm of 600 mg twice a day (BID) (18). On the basis of these findings, the phase 2b/3 program for APL was initiated in treatment-naïve and treatment-experienced HIV-1-infected individuals. During the course of these studies, the clinical development of APL was terminated due to treatment-emergent hepatotoxicity that occurred in a small number of subjects (two individuals with grade 3 or higher hepatotoxicity) receiving APL (24). The reasons for the observed hepatotoxicity with APL are currently unknown; the hepatotoxicity did not appear to be associated with the APL dose.

In the CCR102881 (ASCENT) study, the safety and efficacy of APL plus a fixed-dose combination of lamivudine-zidovudine (3TC-AZT; Combivir) were evaluated in ART-naïve individuals whose plasma virus tested as R5-tropic. Despite the early study termination, protocol-defined virologic failure (VF) was observed in some individuals receiving APL. Following extensive genotypic and phenotypic testing, including clonal analyses of the full-length HIV-1 envelope (env), the predominant characteristic of the circulating virus at the time of VF during APL-3TC-AZT administration was the emergence of the M184V mutation; this was in the absence of any detectable resistance to APL. In addition, the majority of the subjects maintained R5 tropism in plasma, with or without the emergence of the lamivudine resistance mutation M184V.

(This work was presented in part at the XV International Drug Resistance Workshop, Sitges, Spain, 13 to 17 June 2006, and at the Second International Workshop on Targeting HIV Entry, Boston, MA, 20 and 21 October 2006.)

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study population. Informed consent was obtained from all patients or their parent/guardian, and human experimentation guidelines in accordance with GlaxoSmithKline policies and standard operating procedures were followed. Treatment-naïve individuals with no detectable CXCR4-using virus at the screening, who also met the study criteria specified in the protocol, were eligible to enroll in CCR102881 (6). The subjects enrolled in the study could not have major nucleoside reverse transcriptase inhibitor (NRTI) or nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations based on International AIDS Society-defined mutations (16). One hundred forty-five subjects were randomized into the study and received at least one dose of 3TC-AZT BID together with 600 mg APL BID, 800 mg APL BID, or efavirenz once a day.

VF population. VF was defined as (i) incomplete virologic response (the subject did not achieve a 1 log₁₀ c/ml decrease in plasma HIV-1 RNA by week 4 relative to the baseline value) or (ii) virologic rebound (the subject's plasma HIV-1 RNA rebounded to 400 c/ml for two consecutive measurements at least 2 to 4 weeks apart after being previously <400 c/ml on or after week 4, or the subject had two consecutive viral load determinations at least 2 to 4 weeks apart that were >0.5 log₁₀ c/ml plasma HIV-1 RNA from the nadir value of the study, where the nadir is the lowest HIV-1 value that is 400 c/ml on or after week 4).

Clinical samples. Plasma samples for the virologic analyses were collected at screening, day 1, weeks 2 and 4, and every 4 weeks thereafter. The screening samples were analyzed for tropism and the reverse transcriptase (RT) genotype in order to determine eligibility for study participation. For all postscreening samples, tropism determination was attempted when the HIV-1 RNA levels exceeded the validated cutoff for the tropism assay (1,000 c/ml). For the VF samples, tropism, APL susceptibility, RT and protease (PRO) genotype/phenotype, and env clonal analyses were attempted using samples from day 1 and the time of VF.

HIV-1 RT/PRO resistance testing. Plasma samples with documented HIV-1 RNA levels of 500 c/ml were potentially evaluable for RT/PRO resistance testing. HIV pol genotypic data reported were generated either using the Monogram Biosciences (South San Francisco, CA) PhenoSense GT assay (both genotype and phenotype) or the GeneSeq assay (genotype alone). The analyses were focused on and limited to International AIDS Society (16)-defined mutations.

The phenotypic data reported were generated by the Monogram Biosciences PhenoSense GT assay (25). The change (n-fold) in 50% inhibition of viral replication (FCIC₅₀) relative to a drug-sensitive reference virus (NL4-3) was used for determining the sensitivity of each patient's plasma virus to each of the approved antiretroviral drugs.

HIV-1 coreceptor tropism and APL susceptibility. The plasma samples with documented HIV-1 RNA levels of 1,000 copies/ml were potentially evaluable for an analysis of the coreceptor tropism and susceptibility to APL using the Monogram Biosciences PhenoSense HIV entry assay (34). Briefly, in this assay, a pseudovirus is created which contains the env sequence from the sample of interest in the context of a backbone of the remaining sequence of HIV (all but env). The pseudovirus is then used to determine the coreceptor usage in a single cycle assay with a cell line engineered to express CD4 with either CCR5 or CXCR4, the readout being relative light units above the background control. Two measures of susceptibility, the FCIC₅₀, and the maximum calculated percent inhibition (MPI) were generated in the same assay using serial dilutions of the EI. The clinically relevant value for the FCIC₅₀ or MPI has not been determined for APL; however, a threefold FCIC₅₀ was used as the threshold of reduced susceptibility in this analysis, based on the intrinsic assay variability. Of note, phenotypic data for R5X4-tropic clones and DM-tropic populations are obtained for cells that only express CCR5 and therefore should be interpreted with caution.

Clonal analyses of the VFs. Clonal analyses of the full-length HIV-1 env were performed at Monogram Biosciences. Forty-eight envelopes were screened for functionality and tropism. Twelve representative clones underwent more detailed analyses, including tropism confirmation, genotype, and susceptibility to APL. These selected clones represented all of the minority tropic species detected along with some of the majority tropic species. As such, the tropism proportion of the 12 clones may not accurately reflect the relative tropism makeup of the plasma viral quasispecies.

Molecular evolutionary analyses. Homologous nucleotide sequence alignments of full-length env clones were generated for each patient using ClustalX version 1.83 (30) with manual refinement in GeneDoc version 2.6.0.2 (23). Distance- and maximum likelihood (ML)-based phylogenetic reconstructions were performed using PAUP* 4.0b (29), incorporating the best fitting model of sequence evolution and the corresponding values for the rate matrix, shape of the gamma distribution, and proportion of invariant sites as estimated by Modeltest (26). Bootstrap support values were obtained with 100 replicates of ML bootstrapping. One thousand replicates of neighbor-joining bootstrapping were also performed to corroborate the ML findings. The neighbor-joining and ML values were highly concordant, and thus, only the ML trees and bootstrap values are reported.

In order to evaluate nucleotide diversity across time points in the envelope clones from each subject, nucleotide diversity calculations were performed using DnaSP4.10 (28). Nucleotide diversity is defined as the average number of nucleotide differences per site between two sequences drawn at random from the population of sequences (22). If there was a change in env nucleotide diversity over time or a separation of time points on the phylogenetic tree, then statistical tests for genetic differentiation (1, 14, 15) were performed using DnaSP4.10. Due to small sample sizes and in order to be conservative in the determination of the temporal structure, the test statistics were considered significant at a P of <0.001. A rejection of the null hypothesis of no genetic differentiation between subpopulations indicates a partial or complete turnover in the env population between the time points sampled.

Nucleotide sequence accession numbers. The GenBank accession numbers of the sequences reported here are FJ653082 to FJ653313.

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Characteristics of VF in CCR102881. Eight of 145 (6%) subjects met the criteria for protocol-defined VF (Table 1). No subjects in the 3TC-AZT-efavirenz group were identified as having protocol-defined VF prior to the early termination of the study. There was a suggestion of a dose relationship with the number of subjects with VF, six (10%) in the group taking 600 mg APL BID versus two (3%) in the group taking 800 mg APL BID. Most subjects met the protocol definition of VF at or after the week 12 visit (range, 4 to 20 weeks). Seven subjects exhibited virologic rebound, whereas only one subject showed an incomplete virologic response. There was no consistent pattern in either CD4 counts or plasma HIV RNA levels at the time of VF compared with those at day 1.

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TABLE 1. Virologic characteristics of the eight VFs

Population analyses of VFs. M184V was detected in six of the eight subjects with VF (Table 1). There were no other treatment-emergent mutations detected, and the phenotypic results for NRTIs, NNRTIs, and PIs were consistent with the genotypic results (data not shown). Two of the eight subjects with VF had no genotypic or phenotypic evidence of resistance to NRTIs, NNRTIs, or PIs.

All subjects with VF tested as R5-tropic at the population level at both screening and day 1, a time spanning approximately 30 days. Six of the VF subjects had R5-tropic readouts throughout the study, whereas two subjects had DM-tropic readouts at multiple visits after day 1 (Table 1). VF subject N2 had a change in tropism readout to DM-tropic at week 2 through the last visit (week 4 follow-up). Subject N7 had a change in tropism readout to DM-tropic at week 4 and remained DM-tropic until withdrawal (week 16). At withdrawal and at all subsequent visits, no tropism result was obtained for subject N7. Both N2 and N7 had M184V detected, concurrent with changes in population tropism readouts until the time of confirmed VF (data not shown). Of note, changes in the population tropism readout prior to the initiation of therapy (8%) and/or during the course of HIV suppression (<5%) were observed in several subjects not meeting the definition of VF (data not shown).

None of the VF subjects had reduced APL susceptibility (3-fold FCIC₅₀) at the population level (Table 2). In addition, the MPI values at the population level were greater than 98% at both time points for all VF subjects, regardless of the population tropism readout.

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TABLE 2. Clonal analyses of the eight VFs

Clonal analyses of VF subjects. Only R5-tropic clones were detected both at day 1 and at the time of VF in six of the eight subjects with VF (Table 2). The other two subjects had a preexisting R5X4-tropic minority species at day 1 detectable only at the clonal level, despite an R5-tropic population readout. As noted above (Table 1), the population readout in these two subjects changed to DM-tropic after day 1, which was maintained until VF. A clonal analysis demonstrated that the DM-tropic population comprised solely R5X4-tropic clones at the time of VF.

Consistent with the population analysis described above, none of the clones at the time of VF had reduced APL susceptibility (3-fold FCIC₅₀) (Table 2). Subject N5 had one clone detected at day 1 with a 3.67 FCIC₅₀, but there was no evidence for the expansion of this virus at the time of VF. Interestingly, the R5X4-tropic clones at the time of VF from subjects N2 and N7 appeared to be more susceptible to the inhibition of CCR5 usage in the PhenoSense assay than the R5-tropic clones from day 1 for each patient (Fig. 1). The majority of the clones from all VF subjects had MPI values in excess of 99%, with a minority having values in the range of 90 to 95% (Table 2).

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FIG. 1. Susceptibility to APL. APL FCIC50 values for the viral population and env clones were determined in the PhenoSense HIV entry assay. Clonal (diamonds, day 1; squares, VF) and population (circles, day 1; triangles, VF) values are shown for each patient (open for dual-tropic, closed for R5-tropic). Subjects N2 and N7 each had two population values at day 1 due to additional clonal studies.

In order to further understand VF in the context of a lack of resistance to APL, we examined the env nucleotide diversity over time as well as the phylogenetic relationships of full-length env clones from day 1 and time of VF compared to other virologic measures. A decrease in diversity and/or a separation of time points on a phylogenetic tree is indicative of population turnover. Analyses of the VF subjects suggested two evolutionary patterns (Table 3). Five subjects showed no evidence of a change in env nucleotide diversity while maintaining the R5 tropism. These subjects also had env clones intermingled throughout the phylogenetic trees at day 1 and at the time of VF (Fig. 2). Two of these five subjects (N1 and N8) had no evidence of resistance to APL or other study drugs, whereas the other three subjects (N3, N4, and N6) exhibited treatment-emergent M184V.

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TABLE 3. Tropism change and Env nucleotide diversity in the eight VFs

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FIG. 2. Phylogenetic analysis of subject N6. Clonal sequences are designated by the tropism readout in the clonal assay and either day 1 (N6-D1) or VF (week 20, N6-W20). ML phylogeny was reconstructed with support values estimated via 100 replicates of ML bootstrapping. Only bootstrap values of 70% are reported on the tree. Clones remained R5-tropic at VF, occurring coincidently with the detection of the M184V mutation in RT; however, there was no evidence of env population turnover.

A second pattern was seen in three VF subjects (N2, N5, and N7), where there was evidence for a decrease in env nucleotide diversity and a separation of clones across time on the phylogenetic trees. Interestingly, all three subjects had treatment-emergent M184V. Two of these subjects (N2 and N7) exhibited the emergence of an R5X4-tropic quasispecies, detected in a minority of clones prior to the initiation of therapy (Fig. 3). In contrast, subject N5 maintained the R5 tropism, even at the clonal level, yet still exhibited a complete phylogenetic distinction of env clones across time points (Fig. 4). All three subjects with evidence for phylogenetic distinction at the time of VF and a reduction in env nucleotide diversity also had significant population differentiation across time detected by statistical analyses (P < 0.001). Overall, there was no common virologic factor associated with the two evolutionary patterns described above.

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FIG. 3. Phylogenetic analysis of subject N2. Clonal sequences are designated by the tropism readout in the clonal assay and either day 1 (N2-D1) or VF (week 8, N2-W8). ML phylogeny was reconstructed with support values estimated via 100 replicates of ML bootstrapping. Only bootstrap values of 70% are reported on the tree. The change to all R5X4-tropic clones at VF occurred coincidently with the detection of the M184V mutation in RT and was also consistent with env population turnover. R5X4-tropic clones are labeled on the tree ().

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FIG. 4. Phylogenetic analysis of subject N5. Clonal sequences are designated by the tropism readout in the clonal assay and either day 1 (N5-D1) or VF (week 16, N5-W16). ML phylogeny was reconstructed with support values estimated via 100 replicates of ML bootstrapping. Only bootstrap values of 70% are reported on the tree. Clones remained R5-tropic at VF, occurring coincidently with the detection of the M184V mutation in RT and consistent with env population turnover.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The characteristics of the circulating virus in plasma at the time of VF may be impacted by cellular reservoirs, pharmacokinetics/dynamics, viral tropism, genetic diversity of HIV env and/or pol, and other factors. Much is known about the phenotypic and genotypic aspects of resistance to NRTIs, NNRTIs, and PIs. Very little is known about the characteristics of the virus envelope in the context of failure with the standard of care, let alone with regimens containing CCR5 EIs.

The long-term safety and efficacy of CCR5 EI-containing HAART remain to be determined. Several theoretical concerns have arisen with respect to changes in coreceptor tropism and resistance. One concern is whether blocking CCR5 entry will result in a change in coreceptor usage, either by the emergence of a preexisting CXCR4-using (R5X4- and/or X4-tropic) population or by an evolutionary progression from an R5-tropic to a CXCR4-using population. It should be noted at this point that whether or not a change in the population viral tropism in the context of HAART, with or without a CCR5 EI, would have a similar prognostic value for HIV-1 disease progression as seen in natural history studies remains to be determined. Another is the ability to adequately detect the presence of CXCR4-using envelopes in the HIV-1 population both prior to and during antiretroviral therapy (i.e., under various viral load scenarios). A third is the composition of DM-tropic samples from treatment-naïve and -experienced subjects. A fourth, related question surrounds the clinical relevance of the detection of R5X4-tropic quasispecies while on CCR5 EI therapy. Finally, the characteristics of resistance to the CCR5 EI and/or the other components of the regimen may be unique to the particular drug regimen or the patient's treatment history. In order to begin to appropriately address these questions, a careful analysis of samples from clinical trials in both treatment-naïve and treatment-experienced patients is merited.

In CCR102881, no reduced susceptibility to APL was detected at either the population or the clonal level at the time of VF. One possibility for the lack of reduced susceptibility to APL in these VF subjects may be a high genetic barrier for CCR5 EIs. This hypothesis is consistent with in vitro passage studies of APL (17) and other CCR5 EIs (31, 32), which have demonstrated difficulty in generating viruses with high-level resistance. Other reasons for the lack of detectable reduced susceptibility to APL may include drug adherence/tolerability, sampling compartments (plasma versus tissue), and/or assay limitations.

The M184V mutation observed in six of eight subjects with VF was the only treatment-emergent mutation of significance observed in CCR102881. As suggested by Colgrove et al. (5), the M184V mutation may have resulted from the selection of mutants that preexisted therapy at undetectable levels. Consistent with this notion, the three subjects who exhibited env turnover (subjects N2, N5, and N7 [Table 3]) had the M184V mutation detected prior to the time of VF at time points when the HIV RNA values were rebounding toward baseline levels. The high percentage of subjects with the M184V mutation in CCR102881 is similar to preliminary reports of treatment-naïve subjects failing a regimen of 3TC-AZT and the CCR5 EI vicriviroc (12). These data are consistent with the emergence of resistance to the drug with a lower genetic barrier, which is, in these instances, lamivudine.

Two out of the six VF subjects with M184V in CCR102881 had a change in population tropism readout from R5- to DM-tropic concurrent with the detection of the M184V mutation prior to and at the time of confirmed VF. Conversely, four of the six VF subjects with M184V remained R5-tropic at all time points, even at the clonal level. On the basis of this limited amount of data, one could postulate that the virus(es) carrying the M184V mutation at the time of VF may also directly influence the population tropism readout result at the time of VF. As such, the primary step of drug resistance was the selection of the M184V mutation, regardless of the envelope tropism encoded in the same viral genome for that particular quasispecies. This would be true whether or not a change from the pretreatment tropism readout is observed. Consistent with this idea, the concept of pseudohitchhiking has been proposed by Gillespie et al. (10), whereby a genotype in one viral gene product may be associated with a certain genotype in another. Given that HIV pol and env have not been successfully amplified from the same virus genome to date, it is unclear whether a change in tropism is directly associated with the particular viruses bearing the M184V mutation.

Similarly to other biological assays, tropism assays have a threshold of sensitivity. The threshold for the PhenoSense HIV entry assay is accepted as 10% (34), meaning that up to 10 clones out of 100 could be R5X4- and/or X4-tropic and that the assay could call the population R5-tropic. The detailed analyses performed on many subjects from CCR102881 allowed a comparison between population level and clonal-level tropism assays. Of note, the analysis of samples that tested as R5-tropic at the population level prior to therapy (day 1 for both VF subjects and responders) revealed R5X4-tropic clones present as a minority envelope quasispecies in some individuals. Changes in tropism readout were seen in a small percentage of subjects both prior to the initiation of therapy (8%) and while on suppressive therapy with/without a CCR5 EI (<5%) (data not shown). As such, the change in tropism readout in two VF subjects should be interpreted with appropriate caution and in the context of other virologic data from the same individuals.

Turnover in the env population between day 1 and the time of VF was observed in three of the eight subjects who exhibited protocol-defined VF. While all three subjects had the M184V mutation, two showed the emergence of R5X4-tropic clones only and one remained R5-tropic at the clonal level. The remaining five subjects with VF showed no evidence of clonal distinction between day 1 and when VF occurred, regardless of M184V status (three with M184V, two without M184V). As such, env population turnover was not a consistent finding in the context of VF for this study, regardless of M184V status or env tropism at the time of VF compared to day 1. These findings suggest that additional selective pressures may influence the extent of quasispecies diversity during first-line VF with a CCR5 EI-containing regimen.

Protocol-defined VF in CCR102881 was relatively infrequent (4%) (6). Lamivudine resistance in the absence of reduced susceptibility to APL or a change in the viral tropism was the primary characteristic of VF. It should be noted, however, that two factors may have significantly limited our ability to compare the VF of APL-containing regimens with that of a standard of care: (i) the early termination of the studies precluded an analysis of the durability of the virologic response, and (ii) the study was powered to compare the APL-containing arms to each other, and as such, the control arm had fewer patients. It should also be noted that lamivudine resistance was shown in preliminary reports from clinical studies in ART-naïve patients receiving vicriviroc (12) and maraviroc (13). Although the numbers are small, these collective data suggest that a CCR5 EI may require a combination with more potent compounds than a dual-nucleoside backbone for a durable virologic response.

In summary, our data suggest that the clinical utility of viral tropism measurements in the context of VF or virologic response, regardless of whether or not a coreceptor antagonist is part of the drug regimen, remains an open question. Nevertheless, the collective data from CCR102881 are consistent with the hypothesis that combination ART first selects preexisting mutants of HIV-1 that confer the greatest benefit to the virus with the fewest mutations, in this case M184V.

 
We recognize the study subjects for their participation, the clinical investigators, our colleagues at Monogram Biosciences, and the CCR102881 Clinical Study Team. We also thank Joseph Horton for submission of sequences to GenBank.

This study was funded by GlaxoSmithKline.

All authors are current employees of GlaxoSmithKline, with the exception of K.M.K.; however, it should be noted that the work described in this paper was completed before her departure. In addition, she is a consenting author. There are no other potential conflicts of interest.

 
* Corresponding author. Mailing address: GlaxoSmithKline Virology, 5 Moore Drive, Research Triangle Park, NC 27709. Phone: (919) 483-9972. Fax: (919) 315-6787. E-mail: james.f.demarest{at}gsk.com

Published ahead of print on 15 December 2008.

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Antimicrobial Agents and Chemotherapy, March 2009, p. 1116-1123, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01055-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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