JOURNAL OF VIROLOGY, Aug. 2008, p. 8210–8214
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 16
In Vivo Emergence of Vicriviroc Resistance in a Human
Immunodeficiency Virus Type 1 Subtype
Athe M. N. Tsibris,1,2Manish Sagar,2,3Roy M. Gulick,4Zhaohui Su,5Michael Hughes,5
Wayne Greaves,6Mani Subramanian,7Charles Flexner,8Franc ¸oise Giguel,1
Kay E. Leopold,9Eoin Coakley,10and Daniel R. Kuritzkes2,3*
Massachusetts General Hospital, Boston, Massachusetts1; Harvard Medical School, Boston, Massachusetts2; Brigham and
Women’s Hospital, Boston, Massachusetts3; Weill Medical College, Cornell University, New York, New York4;
Harvard School of Public Health, Boston, Massachusetts5; Schering-Plough Research Institute, Kenilworth,
New Jersey6; Human Genome Sciences, Rockville, Maryland7; Johns Hopkins University,
Baltimore, Maryland8; Harvard University, Cambridge, Massachusetts9; and
Monogram Biosciences, South San Francisco, California10
Received 28 February 2008/Accepted 24 April 2008
Little is known about the in vivo development of resistance to human immunodeficiency virus type 1 (HIV-1)
CCR5 antagonists. We studied 29 subjects with virologic failure from a phase IIb study of the CCR5 antagonist
vicriviroc (VCV) and identified one individual with HIV-1 subtype C who developed VCV resistance. Studies
with chimeric envelopes demonstrated that changes within the V3 loop were sufficient to confer VCV resistance.
Resistant virus showed VCV-enhanced replication, cross-resistance to another CCR5 antagonist, TAK779, and
increased sensitivity to aminooxypentane-RANTES and the CCR5 monoclonal antibody HGS004. Pretreatment
V3 loop sequences reemerged following VCV discontinuation, implying that VCV resistance has associated
The human immunodeficiency virus type 1 (HIV-1) enve-
lope third variable loop (V3) is the major structural element of
gp120 that determines coreceptor recognition and specificity
(9, 15, 16). Vicriviroc (VCV; Schering-Plough) and maraviroc
(Selzentry; Pfizer) are allosteric noncompetitive antagonists
that bind to similar sites on CCR5 and antagonize the gp120-
CCR5 interaction (19). To date, data on resistance to these
agents have come largely from in vitro selection studies. Phe-
notypically, resistance manifests as a plateau in the maximum
achievable suppression of viral replication (19). This plateau,
referred to as the percent maximal inhibition, correlates with
viral adaptation to the use of the inhibitor-bound form of
CCR5 for entry (13, 21). Genotypically, VCV resistance has
been associated with a variety of amino-acid-changing muta-
tions throughout the envelope gene (env) that most often in-
volve V3 but whose effect on drug susceptibility depends on the
env backbone into which they are introduced (5). In vitro data
suggest that resistance to the closely related CCR5 antagonist
AD101 does not confer a significant loss of viral fitness (1, 8).
In vivo resistance to the CCR5 antagonists remains poorly
To study the emergence of VCV resistance in vivo, we mon-
itored subjects enrolled in ACTG 5211, a 48-week study of
VCV in 118 HIV-1-infected, treatment-experienced subjects
(3). Among the 90 subjects receiving VCV, we studied all 29
who experienced protocol-defined virologic failure. We ampli-
fied full-length HIV-1 env from plasma samples collected dur-
ing the period from study entry through week 48. These env
sequences were used to generate pseudovirions for examining
VCV susceptibility and coreceptor usage in the PhenoSense
entry susceptibility and Trofile assays (Monogram Bio-
sciences), respectively (20, 22). In 28 of 29 subjects analyzed,
no evidence of decreased VCV susceptibility was observed
(data not shown). Samples from the remaining subject dem-
onstrated increasing VCV resistance over 28 weeks (Fig. 1).
This subject, randomly assigned to receive 10 mg of VCV daily,
experienced protocol-defined virologic failure at week 16 but
continued VCV treatment through week 28 (see Fig. S1 in the
supplemental material). Samples from 13 of the 29 subjects
showed the emergence of CXCR4-using virus at the time of
virologic failure. Virologic failure in the remaining 15 subjects
could not be explained by coreceptor switching or VCV resis-
We assessed the genotypic changes occurring within env over
this same time period by isolating and sequencing multiple
independent full-length clones of env at time points from week
0 through week 48 (Fig. 2). Viral RNA was extracted from the
subjects’ plasma samples by using a QIAamp viral RNA mini
kit (Qiagen), and env amplicons encoding gp160 were gener-
ated by nested PCR as described previously (5). Sequence and
phylogenetic analyses showed that the envelope gene from the
subject with VCV resistance clustered with HIV-1 subtype C
genotypes (data not shown). At weeks 16 and 19, when partial
VCV resistance was observed by the PhenoSense assay, the
* Corresponding author. Mailing address: Section of Retroviral
Therapeutics, Brigham and Women’s Hospital, 65 Landsdowne St.,
Room 435, Cambridge, MA 02139. Phone: (617) 768-8371. Fax: (617)
768-8738. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 21 May 2008.
majority of envelope sequences showed amino acid substitu-
tions K305R, T307I, F316I, T318R, and G319E in the V3 loop
stem (numbering is based upon the HxB2 envelope sequence.
The development of complete phenotypic VCV resistance at
week 28 corresponded to the appearance of the S306P muta-
tion in the V3 loops of all clones. This observation suggests
that the V3 loop mutations present at weeks 16 and 19 confer
partial resistance to VCV, with the addition of S306P leading
to complete resistance. The discontinuation of VCV treatment
at week 28 was associated with a shift toward a VCV-suscep-
tible wild-type virus phenotype by week 48.
We next generated replication-competent recombinant HIV-1
clones to confirm the findings of the population-based data gen-
erated by the PhenoSense assay. We modified a Saccharomyces
cerevisiae gap repair homologous recombination system to create
infectious molecular clones of HIV-1 that carried the predomi-
nant full-length env sequence observed at each time point in an
NL4-3backbone (7) (M. Sagar, unpublished data). Viral stocks
were generated by transfecting 293T cells. The susceptibilities of
the resulting viruses to various HIV-1 inhibitors were assessed on
TZM-bl cells (4, 23). Recombinant viruses expressing the cloned
baseline env sequence were fully susceptible to VCV (Fig. 3a),
whereas those expressing the cloned envelope genes from weeks
16 and 19 demonstrated a progressive decrease in VCV suscep-
tibility. Recombinant virus expressing the cloned week 28 env
showed no inhibition by VCV; increasing VCV concentrations
resulted in increasing virus replication. This observation sug-
gested that fully VCV-resistant virus had adapted to enter cells
with similar findings with a VCV-resistant isolate selected in vitro
(13). Recombinant virus with the cloned week 28 envelope gene
did not infect CXCR4-expressing cell lines (see Table S1 in the
supplemental material), demonstrating that the VCV-resistant
virus continued to use CCR5 exclusively. Viruses expressing env
cloned from the week 48 plasma sample, obtained 20 weeks after
VCV discontinuation, showed restored sensitivity to VCV (Fig.
3a); the week 48 envelopes had near-wild-type V3 sequences.
We next constructed chimeric envelopes by incorporating
FIG. 1. VCV susceptibility of HIV-1 from a subject for whom VCV-containing antiretroviral therapy failed. VCV susceptibility was examined
at week 0 (study entry) (a), week 2 (b), week 8 (c), week 19 (d), week 24 (e), week 28 (f), and week 48 (20 weeks after VCV discontinuation) (g)
by using the PhenoSense entry assay (Monogram Biosciences, South San Francisco, CA) (21). Susceptibilities were plotted as micromolar drug
concentrations versus the percent viral inhibition relative to the infection level in the absence of the drug. The vertical dashed lines indicate the
50% inhibitory concentration for VCV.
VOL. 82, 2008NOTES8211
portions of env from the VCV-resistant week 28 virus into a
VCV-sensitive env backbone (week 0) to ascertain which en-
velope domain(s) determined VCV resistance (Fig. 3b). Dif-
ferent env segments encoding gp120, gp41, V1 to V3, or V3
were amplified using specific primers. These amplified seg-
ments were substituted into the week 0 env by using the mod-
ified yeast gap repair homologous recombination method de-
scribed above. The substitution of the week 28 gp41 sequence
into the week 0 envelope did not alter VCV sensitivity (Fig.
3c). By contrast, recombinant viruses incorporating gp120, the
V1-V3 segment, or the V3 loop from the week 28 envelope
demonstrated complete VCV resistance and enhanced viral
replication in the presence of VCV (Fig. 3d). The substitution
of the V3 loop alone, without V1 or V2, into the VCV-sensitive
week 0 envelope was sufficient to confer complete VCV resis-
tance. Although the V3 loop chimeras included some addi-
tional env sequences from the resistant virus immediately ad-
jacent to both sides of the sequence encoding V3 (Fig. 3b), no
consistent substitutions in these flanking regions were noted.
Therefore, these data support the conclusion that the V3 loop
is the principal determinant of VCV resistance in this virus.
To explore the mechanism of VCV resistance, we tested the
susceptibilities of VCV-resistant virus to other entry inhibitors.
Recombinant viruses expressing the week 28 env showed cross-
resistance to another small-molecule CCR5 inhibitor, TAK779
(2); recombinants expressing the week 48 env showed the res-
toration of TAK779 susceptibility (Fig. 4a). Cross-resistance to
TAK779 and VCV may occur because the two compounds
FIG. 2. Alignment of V3 loop sequences from independent clones
obtained at weeks 0, 16, 19, 28, and 48. The predominant clone at
baseline (week 0) was designated the reference clone. Predicted amino
acid differences are shown, and similarities are indicated with dashes.
The number of independent clones with the same sequence is indi-
cated to the left of each sequence. VF, virologic failure.
FIG. 3. VCV susceptibilities of recombinant viruses with full-length and chimeric envelopes. In each graph, the percentages of inhibition
relative to the viral level in the no-drug control at various inhibitor concentrations are shown. (a) VCV susceptibilities of recombinant viruses with
the predominant full-length envelope at sequential time points after VCV initiation. (b) Schematic representation of the chimeric envelopes; amino
acid numbering is based on the HxB2 reference sequence. (c) VCV susceptibility of recombinant virus expressing a chimeric envelope with week
28 gp41 substituting within the week 0 envelope. (d) VCV susceptibilities of recombinant viruses expressing chimeric envelopes with week 28
envelope segments substituting for week 0 envelope regions. Error bars represent the standard errors of the means of results from two to four
experiments, each performed in triplicate. Nonlinear regression was used to estimate a fitted curve.
8212 NOTES J. VIROL.
occupy similar binding sites on CCR5 (19) and likely lead to
similar allosteric changes in the receptor. By contrast, VCV
resistance sensitized virus to inhibition by the CCR5 monoclo-
nal antibody HGS004 (Human Genome Sciences) and by ami-
nooxypentane (AOP)-RANTES, resulting in 5.4- and 7.8-fold
decreases in 50% inhibitory concentrations, respectively (Fig.
4b and c). HGS004 binds CCR5, competitively antagonizing
gp120 binding, whereas AOP-RANTES triggers CCR5 inter-
nalization (6, 10). Thus, both compounds reduce the number
of CCR5 molecules available on the cell surface. Increased
susceptibilities to HGS004 and AOP-RANTES provided addi-
tional evidence that VCV-resistant virus has a decreased ca-
pacity to utilize the VCV-free form of CCR5. This observation,
coupled with the VCV-enhanced replication of VCV-resistant
virus, suggested that VCV-resistant envelope has decreased
affinity for the non-drug-bound form of the receptor, but for-
mal binding studies are needed to confirm our interpretation
of these data. Previous studies suggested a correlation between
CCR5 affinity and sensitivity to the fusion inhibitor enfuvirtide
(ENF) (14). Interestingly, recombinant viruses with week 0 and
week 28 envelopes showed no difference in ENF sensitivity
(Fig. 4d), suggesting that ENF sensitivity and CCR5 affinity
were not correlated for this viral envelope.
Our results show that in this HIV-1 subtype C isolate from
a VCV-treated subject, increasing VCV resistance was con-
ferred by the stepwise accumulation of mutations on both sides
of the V3 loop stem. The complete loss of VCV susceptibility
correlated with the emergence of S306P at position 11 of the
V3 loop. The presence of a positively charged amino acid
residue at this position is a determinant of CXCR4 usage, but
substitutions such as S306K, S306L, and S306H, which confer
CXCR4 usage, were not found in the samples we analyzed. It
is likely that the introduction of a proline residue at position 11
significantly alters the conformation of the V3 loop. The pres-
ence of a 306P substitution as a naturally occurring polymor-
phism has been reported previously for only two subtype C
viruses (0.05%) and eight subtype B viruses (0.02%) (Los
Alamos Sequence Database [www.hiv.lanl.gov/content/index;
accessed 8 December 2007]). Preliminary data from other sub-
jects experiencing the virologic failure of maraviroc or VCV
show mutations in the V3 loop stem that differ from subject to
subject, although virus from one subject who experienced the
failure of an initial VCV-containing regimen developed
K305R, as seen in the samples from our subject (11, 17). No
signature resistance mutations for the CCR5 antagonists have
been identified to date.
A change in coreceptor usage accounted for the majority of
virologic failures in the phase 3 trials of maraviroc (18) and in
13 of 29 subjects in the present study. The use of CXCR4 by
subtype C viruses appears to be relatively uncommon for rea-
sons that are not yet understood (12). Only two other subjects
enrolled in the parent clinical trial (ACTG A5211) were in-
fected with HIV-1 subtype C virus. One subject was randomly
assigned to the 5-mg VCV arm but discontinued use of the
study drug by week 2 of the study; the other subject was
randomly assigned to the placebo group. Virus from both sub-
jects remained exclusively R5 and VCV susceptible (data not
shown). It is possible that viral or host factors that limit the
emergence of CXCR4-using variants of HIV-1 subtype C con-
strain the pathways available to the virus to escape from VCV
inhibition, thereby favoring the emergence of VCV-resistant
FIG. 4. Resistance to VCV modulates sensitivities to other entry inhibitors. In each graph, percentages of inhibition relative to the viral level
in the no-drug control at various TAK779 (a), HGS004 (b), AOP-RANTES (c), and ENF (d) concentrations are shown. Error bars represent the
standard errors of the means of results from two to four experiments, each performed in triplicate. Nonlinear regression was used to estimate a
VOL. 82, 2008 NOTES8213
mutants that have adapted to use the VCV-bound form of
The loss of virtually all V3 loop mutations by week 48 im-
plies that VCV resistance incurs a fitness cost relative to the
wild-type phenotype in the absence of the drug. This finding
contrasts with published in vitro data (1). Possible explanations
for the disparate results include the limited genetic diversity of
viruses in culture and the absence of immune selective pres-
sure in vitro.
The emergence of VCV resistance in ACTG A5211 was
uncommon, being identified in just 1 of 29 subjects with viro-
logic failure. Although the causal role of V3 loop mutations in
VCV resistance has been demonstrated for this subtype C
virus, additional examples of clinically derived CCR5 antago-
nist-resistant viruses should be studied to elucidate the effects
of the viral subtype on the emergence of VCV resistance and
to describe the full range of changes in env that can confer
CCR5 antagonist resistance.
Nucleotide sequence accession numbers. The nucleotide se-
quences determined in this study have been deposited under
GenBank accession numbers EU664612 to EU664683.
This research was supported by funds from the Clinical Investigator
Training Program, Harvard/MIT Health Sciences and Technology-
Beth Israel Deaconess Medical Center, in collaboration with Pfizer
Inc. and Merck and Co., to A.M.N.T.; NIH grants R37 AI553537 and
K24 RR016482 to D.R.K. and K24 AI-51966 to R.M.G.; and the AIDS
Clinical Trials Group (U01 AI068636), the Harvard Virology Specialty
Laboratory of the AIDS Clinical Trials Group, and the Harvard Uni-
versity Center for AIDS Research (P30 AI060354).
We thank B. M. Baroudy (Schering-Plough) for VCV and the entire
A5211 protocol team for their efforts during this trial. ENF, TAK779,
TZM-bl cells, and pNL4-3 were obtained from the NIH AIDS Refer-
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