Detection of HIV-1 CXCR4 tropism and resistance in treatment experienced subjects receiving CCR5 antagonist—Vicriviroc

Journal of Clinical Virology 55 (2012) 134–139

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Detection of HIV-1 CXCR4 tropism and resistance in treatment experienced subjects receiving CCR5 antagonist—Vicriviroc Paul McNicholas, Regis A. Vilchez 1 , Wayne Greaves, Sushma Kumar, Chinyere Onyebuchi, Todd Black, Julie M. Strizki ∗ Merck Research Laboratories, Kenilworth, NJ, USA

a r t i c l e

i n f o

Article history: Received 3 February 2012 Received in revised form 22 June 2012 Accepted 28 June 2012 Keywords: HIV tropism HIV resistance CCR5 CXCR4 Vicriviroc

a b s t r a c t Background: Vicriviroc (VCV), a small-molecule antagonist of the C–C chemokine receptor 5 (CCR5), blocks HIV’s entry into CD4+ cells. Small studies have suggested that resistance to CCR5 antagonists is slow to develop. Objectives: To examine resistance to VCV in isolates from treatment experienced patients who experienced virologic failure in two phase 3 trials. Study design: Genotypic and phenotypic susceptibility to VCV, and other antiretroviral drugs were evaluated at baseline and at defined intervals during the study. In a post hoc analysis, viral tropism at baseline was evaluated using the Trofile-ES assay. Only subjects with R5-tropic virus were included in the analysis. Viral envelope sequencing was performed on samples from subjects with emergent VCV resistance defined using a relative MPI cutoff. Results: 71/486 subjects treated with VCV for 48 weeks met the protocol-defined virologic failure criteria. 7/71 (10%) had DM/X4 virus at the time of virologic failure; VCV resistance was identified in 4/486 treated subjects (1%). No control subject had detectable DM/X4 virus or VCV resistance at virologic failure. Clonal analysis of envelope sequences from VCV-resistant virus identified 2–5 amino acid substitutions at or near the crown of the V3 loop; however, no signature V3 mutations were identified. Changes outside the V3 loop were also observed in resistant clones; no consistent variant pattern was observed. Conclusions: In these trials, use of a sensitive tropism assay and potent antiretroviral drug combinations contributed to the infrequent detection of X4-tropic virus and VCV resistance. Substitutions in the V3 loop were associated with VCV resistance, however, no specific pattern of amino acid changes were sufficient to reliably predict VCV susceptibility. © 2012 Elsevier B.V. All rights reserved.

1. Background Depending on which coreceptor human immunodeficiency virus 1 (HIV-1) uses to enter CD4 cells, it can be designated as CCR5tropic (R5) or CXCR4-tropic (X4); a dual-tropic virus can use both coreceptors. Because current phenotypic assays cannot distinguish between dual-tropic virus and viral swarms consisting of mixtures of R5 and X4 viruses, isolates that infect CCR5 and CXCR4 expressing cells are designated as dual-mixed (DM) tropic.1 CCR5 antagonists, such as vicriviroc (VCV) and maraviroc (MVC), bind to the coreceptor with high affinity and block entry of

∗ Corresponding author at: Merck & Co., Inc., 770 Sumneytown Pike WP27F-100, West Point, PA 19486, USA. Tel.: +1 215 652 6058; fax: +1 908 823 3065. E-mail addresses: [email protected] (R.A. Vilchez), [email protected] (J.M. Strizki). 1 Present address: Global Pharmaceutical Research and Development, Abbott Laboratories, USA. 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.06.021

R5-tropic viruses through allosteric interactions.1–4 Because of the allosteric nature of the CCR5 antagonists, phenotypic resistance development is manifested not as a large increase in the drug concentration required to inhibit virus replication by 50% (IC50 ) value but rather as a decrease in the maximum achievable level of inhibition, also termed maximum percent inhibition (MPI), of viral entry. This MPI parameter appears to be a reliable marker for identifying viruses with reduced susceptibility to CCR5 antagonists.4–8 2. Objectives Studies have suggested that resistance to coreceptor CCR5 antagonists is slow to develop and may involve multiple amino acid changes in the viral envelope gene.2,4–11 In the present study, we analysed genotypic and phenotypic HIV-1 tropism and drugresistance in isolates from treatment experienced patients with virologic failure in the VCV phase 3 trials to identify and characterize the features of resistance to CCR5 antagonists.

P. McNicholas et al. / Journal of Clinical Virology 55 (2012) 134–139

3. Study design 3.1. Study population The design of the VCV phase 3 trials was previously described.12 Briefly, 2 identical double-blind multicenter phase 3 studies, VICTOR-E3 (NCT00523211) and VICTOR-E4 (NCT00474370), among treatment experienced subjects compared VCV with placebo, each in combination with optimized background therapy (OBT). Both studies were conducted in accordance with principles of good clinical practice and were approved by the appropriate institutional review boards and regulatory agencies. Investigators selected OBT based on results of the PhenoSense GT test (Monogram Biosciences, San Francisco, CA) and according to the International AIDS Society—USA guidelines.13 Only subjects with R5-tropic virus at baseline by the standard Trofile assay (Monogram Biosciences) were randomized for treatment.14 A retrospective reanalysis of HIV-1 tropism was performed using the more sensitive Trofile-ES assay (Monogram Biosciences) before the unblinding of the week 48 results.15 Subjects confirmed to have R5 virus by both tropism assays were included in the primary efficacy evaluation population which consisted of 486 and 235 subjects in the VCV and control groups, respectively. Virological failure was defined as failure to achieve a 0.5-log10 reduction in HIV-1 RNA from baseline to at least week 8, with confirmation within 4 weeks; increase of three or more times the baseline HIV-1 RNA level at the week 2 visit or thereafter, with confirmation within 4 weeks, or increase in HIV RNA by ≥1.0 log10 from the nadir value, with confirmation within 4 weeks; or rebound in HIV-1 RNA to >1000 copies/mL on two consecutive occasions (≥2 weeks apart) in subjects previously confirmed to have undetectable levels. 3.2. HIV tropism determinations HIV tropism and VCV susceptibility were assayed using the standard Trofile and PhenoSense Entry assays at screening, baseline, and at weeks 2, 12, 20, 32, and 48 or study discontinuation for subjects with HIV RNA > 500 copies/mL. IC50 and MPI values were calculated from dose–response curves as previously described.5,6 VCV susceptibility was also expressed as relative MPI (R-MPI), and fold change IC50 (FC-IC50 ) calculated as the ratio of subject and control virus values.

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Table 1 Emergent resistance to drug classes in the OBT of subjects with virologic failure. Drug class

Nucleoside/nucleotide RT inhibitors Protease inhibitors Integrase inhibitor Total subjects with OBT resistance

Subjects OBT resistance on study, n (%) VCV (n = 71)

Control (n = 38)

15 (21) 3 (4) 2 (3) 20 (28)

7 (18) 3 (8) 1 (3) 11 (29)

Abbreviations: OBT, optimized background therapy; VCV, vicriviroc.

novo resistance to drugs in the nucleotide/nucleoside reverse transcriptase inhibitors class was most common and occurred at similar rates in both treatment groups (Table 1). Resistance to raltegravir was uncommon and was detected in only 2 subjects in the VC group and 1 subject in the control group. 4.2. HIV-1 tropism and virologic failure Overall, DM- and/or X4-tropic virus was detected infrequently (7/71, 10%) among subjects with PDVF in the VCV group (Table 2). The majority of subjects with virologic failure had only R5-tropic virus detected at the time of failure and had 3 or more fully active drugs in their OBT. None of the subjects with less than 2 active drugs in their OBT had DM/X4 virus detected on study. 4.3. Determining cutoff values for VCV resistance Baseline samples were tested to determine the range of susceptibility of VCV. In addition, a control virus, HIV JrCSF, was tested in parallel for each assay run to control for variability. The baseline MPI values exhibited a relatively wide range (73–100%, mean 97%) of susceptibility to VCV. However, when these values were compared with the JrCSF control run in parallel, a similar wide range of MPI values was observed (81–100%, mean 93%) demonstrating that inherent variability in the MPI existed from assay to assay (Supplemental Data, Fig. 1). Therefore, to normalize the subject MPI values and control for the day-to-day assay variability, we calculated the R-MPI value. Resistance to VCV was defined as an R-MPI value that was 2 standard deviations below the mean value for all baseline isolates. The R-MPI values were 0.94 and 0.95 for samples in the VICTOR-E3 and VICTOR-E4 trials, respectively. 4.4. Baseline resistance to VCV

3.3. Clonal analysis of HIV gp160 For VCV-resistant virus, 12 randomly selected gp160 clones per time point from baseline and on-treatment samples were sequenced and tested for susceptibility to VCV. In addition, genotypic tropism assessments were made using a Web-based position sequence scoring matrix (PSSM) algorithm (http://indra.mullins. microbiol.washington.edu/webpssm/). Alignment of gp160 sequences was performed using CLUSTAL 2.0.8 multiple sequence alignment software. Consensus V3 loop sequences were generated using Vector NTI 9.0 software. 4. Results 4.1. Virologic failure and the emergence of resistance to drugs in the OBT The proportion of subjects with virologic failure in the VCV group and in the control group were 15% (71/486) and 16% (38/235), respectively. Among subjects with virologic failure, the proportion with emergent resistance to ≥1 of the drugs in their OBT was similar in the VCV and control group (20/71, 28% vs. 11/38, 29%). De

Based on the criteria described above, 3 subjects (1305, 6801, and 6829) had virus with reduced VCV susceptibility at baseline as determined by R-MPI values below the resistance cutoff (Supplemental Data, Table 1). However, none of these 3 subjects had confirmed VCV resistance following initiation of therapy and therefore were excluded from further analysis. Two subjects, 1305 and 6829, achieved complete viral suppression by week 1 and week 20, respectively, although subject 1305 discontinued early because of an adverse event. Subject 6801, failed to achieve full viral suppression on study and discontinued by week 24. However, ontreatment R-PMI values from this subject did not meet the criteria for VCV resistance. 4.5. Emergent resistance to VCV on study Using the R-MPI cutoff values, we identified the emergence of VCV-resistant viruses in 4 (1%) of 486 subjects in the VCV treatment group (Table 3). All 4 subjects experienced virologic failure. Three subjects were infected with subtype B virus, and 1 subject (6267) had subtype F1 virus. Dose–response curves for individual gp160 clones from baseline and on-treatment samples from each of these

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P. McNicholas et al. / Journal of Clinical Virology 55 (2012) 134–139

Table 2 HIV-1 tropism at time of virologic failure by baseline overall sensitivity score. Subjects with virologic failure at week 48, n (%) VCV (n = 71) Tropism at VF/Baseline OSS

<2 (n = 2)

CCR5 CXCR4 Dual/mixed Not reportable

2 0 0 0

Control (n = 38) 2 (n = 13) 10 (77) 2 (15) 0 1 (8)

≥3 (n = 55) 46 (84) 2 (4) 3 (5) 4 (7)

<2 (n = 4)

≥3 (n = 22)

2 (n = 12)

4 (100) 0 0 0

11 (92) 0 0 1 (8)

22 (100) 0 0 0

Tropism assay was not performed for any subject with HIV RNA < 500 copies/mL. Abbreviations: OSS, overall sensitivity score; VF, virologic failure; VCV, vicriviroc.

subjects demonstrated a reduced plateau (MPI) and R-MPI values below the set cutoffs (Fig. 1). Phenotypic resistance to VCV was detected after 20 weeks of therapy in subjects 6308 and 6242 and after 32 weeks in subject 6267. For subject 1803, VCV resistance was first detected at week 12 and there was further loss of susceptibility at weeks 20 and 48. For all 4 subjects, the VCV IC50 values determined for resistant isolates were either similar to or lower than the values measured for the susceptible baseline samples. Virus from subjects 6308, 6242, and 6267 was R5-tropic at all time points tested. In contrast, virus from subject 1803 was classified as R5 at baseline and week 48 but DM-tropic at weeks 12 and 20. 4.6. Clonal analysis of HIV-1 gp160 sequences in subjects with phenotypic resistance to VCV A summary of V3 loop sequences, tropism, and VCV susceptibility determinations for the clonal analysis is shown in Table 4. Changes observed in resistant clones identified across the entire gp160 for each subject are summarized in Supplemental Data, Fig. 2. As with the V3 analysis, no consistent pattern of non-V3 mutations was identified that correlated with phenotypic resistance to VCV. A detailed analysis of the results for each subject follows: Subject 6308. All 12 baseline clones had R-MPI values >0.94; 11 and 10 clones from weeks 20 and 32 had R-MPI values <0.94, respectively (Table 4). Alignment of the 12 baseline clones with the

phenotypically resistant clones from weeks 20 and 32 revealed a number of conserved changes. The only amino acid change in gp120 that was specific to all 11 week 20 clones was F317V. Outside of the V3 region, the following conserved mutations were noted: R344K, K/T362N, N386S, and D461N/S. In gp41, a T4M mutation was seen in week 20 and 32 clones but was absent at baseline (data not shown). Interestingly, 9 of 12 clones isolated from the week 20 sample were determined to be DM although the virus swarm was classified as R5. In addition, 8 of these resistant DM clones shared the same V3 loop sequence as one R5 tropic clone from the same sample. Subject 6242. At baseline and week 24, 8 and 10 clones exhibited R-MPI values >0.94 and <0.94, respectively. From an alignment of the 8 susceptible and 10 resistant clones the only amino acid variant in gp120 that was specific to all 10 resistant clones was H/Y308P alone or in combination with K305R and/or G313R. Interestingly, the 2 clones containing a R336G variant were fully susceptible to VCV, despite having variants at K305R, H308P, and G313R. Subject 6267. All 12 baseline clones were susceptible to VCV and 7 of the week 43 clones were resistant to VCV. An alignment of susceptible and resistant clones did not identify any amino acid changes in gp120 that were specific to all 7 week 43 clones. As observed in subject 6308, several (4/12) week 43 clones were determined to be DM, although the viral pool was R5-tropic and 3 of 4 DM clones had the same V3 loop sequence as R5-tropic clones. Several clones with the same V3 loop showed a wide range of susceptibility to VCV (R-MPI, 0.66–1.0).

Table 3 Characteristics of subjects with emergence phenotypic VCV resistance. OSSa

Tropismb

CD4 count (␮L)

0 12 20 32

3

R5 R5 R5 R5

193 180 240 281

6242/B

0 12 20 24

3

R5 R5 R5 R5

6267/F1

0 12 20 32 43

1803/B

0 2 12 20 48

Subject/HIV subtype

Time point (weeks)

6308/B

IC50 (nM)c

Fold change IC50 d

MPI (%)c

Relative MPIe

536,000 85,800 218,000 594

3.21 3.11 2.88 3.29

0.79 0.79 1 0.79

99 96 84 90

1 1 0.86 0.93

356 423 301 453

10,400 1380 26,000 41,400

2.89 1.89 2.26 2.44

0.59 0.6 0.72 0.35

92 90 86 76

1 0.97 0.93 0.81

2

R5 R5 R5 R5 R5

252 139 156 170 231

235,000 33,600 53,300 50,500 18,000

5.15 2.88 1.58 2.02 2.73

0.4 0.51 0.59 0.48 0.44

97 88 92 89 65

1.1 0.96 0.96 0.93 0.69

1

R5 R5 DM DM R5

56 219 215 246 231

1,520,000 830,000 58,800 54,600 85,300

4.76 4.17 2.91 3.85 1.40

0.96 0.85 0.59 0.78 0.53

94 96 83 58 63

1 1 0.89 0.62 0.65

Viral RNA (copies/mL)

IC50 , half maximal inhibitory concentration; MPI, maximum percent inhibition, OSS, overall susceptibility score; VCV, vicriviroc. a OSS determined at screening. b Results are for the viral swarm using the Trofile assay. c Values are for the viral swarm using PhenoSense Entry assay. d IC50 of subject/IC50 of virus control evaluated in parallel. e MPI of subject/MPI of virus control evaluated in parallel.

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Fig. 1. VCV dose–response curves for pseudotype viruses generated from gp160 clones isolated from baseline and resistant viral populations.

Subject 1803. All 12 baseline and week 48 clones exhibited R-MPI values >0.98 and <0.69, respectively. An alignment of the baseline and week 48 clones identified several amino acid changes in gp120 (T133N, K/R168Q, E172K, D/N178I, R185V, D186N, L201I, K/T305N, A316V, K440Q) and 1 in gp4 (T4M) that were specific to all 12 week 48 clones. In addition, 10 of these 12 clones were DM-tropic. 5. Discussion This investigation of subjects infected with HIV-1 CCR5-tropic as defined by the Trofile-ES assay showed that detection of DM or X4-tropic virus and emergent resistance to VCV were infrequent in subjects failing a VCV-containing regimen. This is in contrast to the results in the MVC trials, where DM or X4 virus was detected in 57%

of treatment failures.16 The difference between the VCV and MVC trials is likely due to differences in the sensitivity of the tropism assays used for screening and treatment factors. In the MVC trials, tropism at baseline was assessed with the original, less sensitive Trofile assay compared with the enhanced sensitivity Trofile-ES assay used in the VCV trials to exclude subjects with pre-existing DM/X4 virus at baseline.16,17 Therefore, it is likely that a larger number of subjects had low level X4-tropic virus present at baseline in the MVC trials that would have become detectable after initiation treatment. The number of subjects with 3 or more fully active drugs in the OBT was higher in the VCV trials compared with the MVC trials (55% vs. 32%).12,16 Thus, more potent OBT regimens may have suppressed outgrowth of pre-existing minor X4-tropic variants.

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P. McNicholas et al. / Journal of Clinical Virology 55 (2012) 134–139 Table 4 VCV phenotypic susceptibility and V3 sequences of gp160 clones from baseline and on-study samples from subjects with VCV resistance.

ID

6308

6242

6267

1803

Time

# clones

Tropism

Baseline

11 1

R5 R5

MPI (range) (%) 97–100 100

Week 20

1 1 8 1

R5 R5 DM DM

Week 32

4 3 5

Baseline

R-MPI (range)

V3 loop sequence

1.0 1.0

CTRPNNNTRRSISIGPGRAFYTTGDIIGDIRQAHC .........K..........V..............

84 94 66–87 82

0.88 0.80 0.76–0.97 0.85

...................V............... ............G......V............... ............G......V............... ..........R.G......V...............

R5 R5 DM

89–100 76–86 68–77

0.87–1.0 0.78–0.89 0.71–0.80

................................... ............G......V............... ............G......V...............

7 1 1 1 2

R5 R5 R5 R5 R5

84–100 100 97 96 76–78

0.88–1.0 1.1 1.0 1.0 0.81–0.82

CTRPNNNTRKSIHIGPGGAFYATGDIIGDIRQAHC ....................D.............. ..............D.................... ............Y...................... ............P....R.................

Week 24

3 1 2 1 1 4

R5 R5 R5 R5 R5 R5

75–86 100 58–66 40 100 56–69

0.79–0.91 1.1 0.62–0.70 0.42 1.1 0.59–0.73

............P....R................. ............P....R............G.... .........R..P....R................. .........R..P...................... .........R..P.................G.... ............P......................

Baseline

4 1 2 2 2 1

R5 R5 R5 R5 R5 R5

98–100 98 95–99 96–98 95–98 98

1.0–1.1 1.0 1.0 1.0 1.0 1.0

CTRPNNNTRKSIHITPGRTFFTGDIIGDIRQAHC ..................A................ ...................L............... .................KA................ ..............S...AV............... ............N.S...AV...............

Week 43

1 1 1 1 3 3 1 1

R5 DM R5 R5 R5 DM R5 R5

64 74 66 64 62–95 73–99 100 68

0.67 0.87 0.67 0.68 0.66–1.0 0.77–1.0 1.1 0.72

..........G.........F.............. .............L......I.............. ....................I.............. ....................I........M..... ............N.A...A.I.............. ............N.A...A.I.............. ...S........N.A...A.I.............. ............N.A...A.I....L.........

Baseline

3 5 1 1 1 1

R5 DM R5 R5 R5 R5

94–100 93–97 94 97 100 98

0.99–1.1 0.98–1.0 0.99 1.0 1.0 1.0.

CTRPNNNTRKGIHIGPGRAFYATDIIGDIRQAHC .................................. ...................L.............. .........T........................ .....................G............ ..........S..........G............

Week 48

1 3 1 6 1

R5 DM R5 DM DM

95 53–64 66 51–64 53

1.0 0.56–0.68 0.69 0.53 0.56

.........N........V............... .........N........V............... .........N.......KV............... .........N.......KV............... .........N.......KV......V........

As CCR5 antagonists are a relatively new drug class, cutoff values for susceptibility have not been firmly established. In addition, CCR5 antagonists are allosteric inhibitors, and resistance is most reliably measured as a decrease in the MPI or R-MPI parameter rather than a shift in IC50 concentration. In the MVC trials, an MPI < 95% was used as a cutoff to identify MVC resistant isolates.18 However, in the VCV studies, considerable variability was noted in the MPI values at baseline. Similar MPI variability was also observed for the assay control virus and appeared to correlate with fluctuations in the MPI values of subject isolates. Therefore, to better control for day-to-day assay variability, an R-MPI value was calculated. Using this approach, we were able to distinguish between low MPI values caused by assay variability and true VCV resistance.

One possible explanation for the relative difference in the spread of baseline MPI values reported for MVC and VCV is the range of drug concentrations used for the individual drugs in the PhenoSense assay. For MVC, the highest drug concentration used is 3000 nM compared with 667 nM for VCV. This difference in drug concentrations, which defines MPI value, likely accounts for the increased variability of VCV compared with MVC. No consistent pattern of resistance-associated variants was identified in these studies. The findings are consistent with earlier VCV studies where no signature V3 loop mutations were identified.5–8 These data suggest that other regions in the envelope, outside of V3, play a key role in V3 loop conformation and interact directly with the CCR5 coreceptor. Recently, Berro et al.19

P. McNicholas et al. / Journal of Clinical Virology 55 (2012) 134–139

performed a meta-analysis of published amino acid substitutions identified in VCV and MVC resistant viruses to identify patterns of V3 substitutions associated with resistance; the most common V3 substitutions were residues K10, H13, G24 and D25. None of the 4 subjects identified with VCV resistance virus isolates and analyzed in these studies had substitutions at positions G24 or D25. Two VCV resistance virus isolates had changes at position 10; and three of the four VCV resistance isolates had changes at position 13 in the crown of the V3-loop. However, the presence of substitutions at these positions alone could not explain the differences in the viral susceptibility to VCV in pseudoviruses expressing these clones. The variability and complexity of the HIV-1 envelope and its interaction with the cellular coreceptor pose challenges to the development of a predictive genotypic model for CCR5 antagonist resistance. In conclusion, the use of a sensitive tropism assay at baseline and potent OBT regimens likely contributed to the infrequent detection of HIV-1 DM or X4-tropic virus and emergent resistance to VCV in treatment experienced patients. Additional genotypic and phenotypic data from clinical studies will be needed to develop better assays for quantifying resistance and defining cutoffs to predict treatment response with CCR5 antagonists. Authors’ contribution In this study, Paul McNicholas and Julie M. Strizki have done data analysis, figure generation, and manuscript writing. The data analysis and manuscript writing works were shared by Regis A. Vilchez, who did study design too and by Todd Black, who conducted the discussion. With them, Wayne Greaves too rendered study design and manuscript editing. Sushma Kumar and Chinyere Onyebuchi have done study execution and data collection. Funding These studies were supported by Schering-Plough Corp., which is now a part of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Whitehouse Station, NJ, USA. Competing interests Paul McNicholas and Regis A. Vilchez were employees of Schering-Plough/Merck. Wayne Greaves, Sushma Kumar, Chinyere Onyebuchi, Todd Black and Julie M. Strizki are employees of Schering-Plough/Merck. Ethical approval The VICTOR-E3 (NCT00523211) and VICTOR-E4 (NCT00474370) studies were conducted in accordance with principles of good clinical practice and were approved by the appropriate institutional review boards and regulatory agencies. Acknowledgments The authors would like to thank Dr. Jacqueline Reeves and Dr. Wei Huang at Monogram Biosciences for providing dose–response graphics and for helpful discussions on phenotypic data interpretation. In addition, the authors thank all the participants and

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investigators who took part in the VICTOR-E3 and VICTOR-E4 trials. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2012.06.021. References 1. Dorr P, Westby M, Dobbs S, Griffin P, Irvine B, Macartney M, et al. Maraviroc (UK427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother 2005;49:4721–32. 2. Kuhmann SE, Pugach P, Kunstman KJ, Taylor J, Stanfield RL, Snyder A, et al. Genetic and phenotypic analyses of human immunodeficiency virus type 1 escape from a small-molecule CCR5 inhibitor. J Virol 2004;78:2790–807. 3. Strizki JM, Tremblay C, Xu S, Wojcik L, Wagner N, Gonsiorek W, et al. Discovery and characterization of vicriviroc (SCH417690), a CCR5 antagonist with potent activity against human immunodeficiency virus type 1. Antimicrob Agents Chemother 2005;49:4911–9. 4. Westby M, Smith-Burchnell C, Mori J, Lewis M, Mosley M, Stockdale M, et al. Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry. J Virol 2007;81:2359–71. 5. McNicholas P, Wei Y, Whitcomb J, Greaves W, Black TA, Tremblay CL, et al. Characterization of emergent HIV resistance in treatment-naive subjects enrolled in a vicriviroc phase 2 trial. J Infect Dis 2010;201:1470–80. 6. McNicholas PM, Mann PA, Wojcik L, Phd PQ, Lee E, McCarthy M, et al. Mapping and characterization of vicriviroc resistance mutations from HIV-1 isolated from treatment-experienced subjects enrolled in a phase II study (VICTOR-E1). J Acquir Immune Defic Syndr 2011;56:222–9. 7. Ogert RA, Wojcik L, Buontempo C, Ba L, Buontempo P, Ralston R, et al. Mapping resistance to the CCR5 coreceptor antagonist vicriviroc using heterologous chimeric HIV-1 envelope genes reveals key determinants in the C2-V5 domain of gp120. Virology 2008;373:387–99. 8. Pugach P, Marozsan AJ, Ketas TJ, Landes EL, Moore JP, Kuhmann SE. HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry. Virology 2007;361:212–28. 9. Ogert RA, Hou Y, Ba L, Wojcik L, Qiu P, Murgolo N, et al. Clinical resistance to vicriviroc through adaptive V3 loop mutations in HIV-1 subtype D gp120 that alter interactions with the N-terminus and ECL2 of CCR5. Virology 2010;400:145–55. 10. Gulick RM, Su Z, Flexner C, Hughes MD, Skolnik PR, Wilkin TJ, et al. Phase 2 study of the safety and efficacy of vicriviroc, a CCR5 inhibitor, in HIV-1-infected, treatment-experienced patients: AIDS clinical trials group 5211. J Infect Dis 2007;196:304–12. 11. Suleiman J, Zingman BS, Diaz RS, Madruga JV, DeJesus E, Slim J, et al. Vicriviroc in combination therapy with an optimized regimen for treatment experienced subjects: 48 week results of the VICTOR-E1 phase 2 trial. J Infect Dis 2010;201:590–9. 12. Caseiro MM, Nelson M, Diaz RS, Gathe J, De Andrade Neto JL, Slim J, et al. Vicriviroc plus optimized background therapy for treatment-experienced patients with CCR5 HIV-1 infection: final results from two randomized trials. J Infect 2012, http://dx.doi.org/10.1016/j.jinf.2012.05.008. 13. Hammer SM, Saag MS, Schechter M, Montaner JS, Schooley RT, Jacobsen DM, et al. Treatment for adult HIV infection. JAMA 2006;296:827–43. 14. Whitcomb JM, Huang W, Fransen S, Limoli K, Toma J, Wrin T, et al. Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 coreceptor tropism. Antimicrob Agents Chemother 2007;51:566–75. 15. Reeves JD, Coakley E, Petropoulos CJ, Whitcomb JM. An enhanced-sensitivity Trofile HIV coreceptor tropism assay for selecting patients for therapy with entry inhibitors targeting CCR5: a review of analytical and clinical studies. J Viral Entry 2009;3:94–102. 16. Fätkenheuer G, Nelson M, Lazzarin A, Konourina I, Hoepelman AI, Lampiris H, et al. Subgroup analyses of maraviroc in previously treated R5 HIV-1 infection. N Engl J Med 2008;359:1442–55. 17. Gulick RM, Lalezari J, Goodrich J, Clumeck N, DeJesus E, Horban A, et al. Maraviroc for previously treated patients with R5 HIV-1 infection. N Engl J Med 2008;359:1429–41. 18. Mori J, Mosley M, Lewis M, Simpson P, Toma J, Huang W, et al. Characterization of maraviroc resistance in patients failing treatment with CCR5-tropic virus in MOTIVATE 1 and MOTIVATE 2. Antiviral Ther 2007;12:S12. 19. Berro R, Klasse PJ, Moore JP, Sanders RW. V3 determinants of HIV-1 escape from the CCR5 inhibitors maraviroc and vicriviroc. Virology 2012;427:158–65.