Broad Phenotypic Cross-Resistance to Elvitegravir in HIV-Infected
Patients Failing on Raltegravir-Containing Regimens
Carolina Garrido,aJorge Villacian,bNatalia Zahonero,aTheresa Pattery,bFederico Garcia,cFelix Gutierrez,dEstrella Caballero,e
Margriet Van Houtte,bVincent Soriano,aand Carmen de Mendozaaon behalf of the SINRES Group
Hospital Carlos III, Madrid, Spaina; Virco BVBA, Mechelen, Belgiumb; Hospital Clínico Universitario San Cecilio, Granada, Spainc; Hospital General Universitario, Elche, Spaind;
and Hospital Vall d’Hebrón, Barcelona, Spaine
patients, with dramatic improvements in survival. However,
selection for drug resistance still represents a major challenge
and promotes switches in therapeutic regimens for a substan-
tial proportion of patients (3). Virologic failure with some an-
tiretroviral drugs is associated with cross-resistance to agents
within the same family, limiting rescue therapeutic options
(16). Nevirapine and efavirenz are good examples of cross-
resistance between members of the same drug family, almost
completely sharing resistance patterns. To a lesser extent, this
also occurs with failures of some protease inhibitors (12) and
ily for the treatment of HIV infection. Raltegravir (RAL) is the
first-in-class approved drug for clinical use in both treatment-
vir (EVG) will most likely be the second INI compound to be
marketed (4). Resistance to INIs has been shown to be driven by
changes located mainly in the central domain of the viral inte-
grase. The results of clinical trials and clinical experience have
highlighted that failure on RAL-containing regimens is generally
driven by the selection of the mutations Y143RHC, Q148HRK,
and/or N155H, often accompanied by secondary changes
(G140SA, E138K, and L74M, etc.) (2). However, a relatively high
tance mutations might exist, a recent report highlighted that the
absence of INI resistance mutations in these individuals is ex-
he introduction of highly active antiretroviral therapy
(HAART) has transformed the prognosis of HIV-positive
plained mainly by poor drug compliance, given that RAL plasma
levels were undetectable in most of them (6).
Information about resistance to EVG in vivo is scarce. In the
phase II GS-US-183-105 study (13), 28 individuals failed EVG
after 24 weeks of treatment. Overall, 39% of them selected the
E92Q mutation, 32% selected the Q148HRK mutation, and 25%
selected the N155H mutation. Accordingly, broad cross-resis-
tance between RAL and EVG should be expected. The aim of our
study was to characterize RAL phenotypic susceptibilities in sam-
ples from a group of patients experiencing RAL failure, exploring
whether mutations not previously involved in RAL resistance
MATERIALS AND METHODS
viral regimen were identified during 2009. Virological failure was defined
as the first sample with plasma HIV RNA levels of ?50 copies/ml, con-
firmed with a second specimen collected within a month. Plasma speci-
mens collected at the time of the first RAL failure were then sent to Hos-
Received 21 December 2011 Returned for modification 16 January 2012
Accepted 15 March 2012
Published ahead of print 26 March 2012
Address correspondence to Vincent Soriano, email@example.com, or
Carolina Garrido, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
June 2012 Volume 56 Number 6Antimicrobial Agents and Chemotherapyp. 2873–2878aac.asm.org
pital Carlos III along with information on viral load, CD4 counts, and
were also sent to be used as controls. Most participating centers belonged
to RIS (Red de Investigación en SIDA), the government-funded Span-
ish AIDS research network, which involves around 25 HIV clinics
across the country and whose main characteristics were described else-
where previously (17). All specimens that were examined in the cur-
rent study were part of the repository collected for the SINRES study,
a previous survey that characterized RAL failures virologically in a
total of 106 patients (6). Signed informed consent was obtained from
was performed by bulk sequencing using an in-house PCR protocol with
primers and conditions previously described (7). Primary integrase resis-
tance mutations were defined as those changes that caused a significant
loss of drug susceptibility by themselves. This was the case for the
Y143RHC, Q148HRK, and N155H mutations.
Phenotypic resistance analysis. Viral RNA was isolated from all
plasma samples by using NucliSENS EasyMAG (bioMérieux). One am-
plicon per sample containing the reverse transcriptase-integrase (RT-IN)
viruses were titrated and subjected to an antiviral experiment with MT4-
LTR-eGFP cells, as previously described (20). Briefly, MT4-LTR-eGFP
cells were inoculated with a titrated amount of virus in the presence of
3-fold dilutions of the compound tested. After 3 days of incubation at
suring HIV Tat-induced enhanced green fluorescent protein (eGFP) ex-
pression. Using HIV-1 wild-type strain IIIB as a reference, fold change
(FC) values were calculated by dividing the mean 50% effective concen-
tration (EC50) for a recombinant virus stock by that for the reference
a clonal database (991 clones from 153 clinical isolates) as the 97.5th
percentile of the log FC phenotypes of patient-derived clonal viruses
not containing any of the mutations listed in the RAL package insert
label, including 74M, 92Q, 97A, 138A/K, 140A/S, 143C/H/R, 148H/
K/R, 151I, 155H, 163R, 183P, 226C/D/F/H, 230R, and 232N, and/or
not containing mutations with score of ?0 for RAL-EVG by Stanford
algorithm 6.0.11. Before the BCO was calculated, outliers (log FC ?
mean log FC plus 3 standard deviations) were removed (22). Thus,
biological cutoffs were preliminarily established as an FC of 2 for RAL
and an FC of 1.9 for EVG.
Statistical analysis. Results are expressed as medians (interquartile
parametric tests using Mann-Whitney or chi-square tests. FC values were
bivariate analysis. All statistical analyses were carried out by using SPSS
v15 software (SPSS Inc., Chicago, IL).
Nucleotide sequence accession numbers. All sequences examined in
this study have been submitted to the GenBank database under accession
numbers JQ716857 to JQ716918.
Study population. A total of 61 samples were examined: 21
belonging to INI-naïve patients and 40 obtained from patients
experiencing early virological failure on RAL-containing regi-
mens. All INI-naïve individuals were antiretroviral experi-
enced. The main characteristics of this population are summa-
rized in Table 1.
Resistance testing for INI-naïve patients. Phenotypic results
that all but 1 (95.2%) were susceptible to RAL. The single sample
displaying an FC of 2.4 for RAL did not harbor any primary or
secondary known integrase resistance mutation. However, com-
pared to reference sequences, the following changes in the inte-
samples displayed an FC above 1.9 for EVG. Neither primary nor
secondary resistance changes were found in these specimens, as
shown in Table 2.
Resistance testing for RAL failures. Overall, 11 out of 40
(27.5%) samples collected from patients experiencing early RAL
failure harbored primary INI resistance mutations, including the
and a complex pattern including the Y143HY and N155H muta-
tions (n ? 1).
tations. Three samples, which did not contain any primary INI
resistance change, displayed low FC values for RAL (2, 2.5, and
26.5 to 125.3) for the 11 patients with INI resistance mutations.
The main characteristics of these patients are reported in Table 3,
of drug adherence.
failures displayed impaired EVG sensitivity. Overall, the median
to 99.93] versus 2.45 [IQR, 2.36 to 2.83]; P ? 0.001). Table 3
reports the genotypic and phenotypic data for EVG in these pa-
both RAL and EVG although with slight differences. The Y143R
mutation conferred a higher level of resistance to RAL than to
EVG, with the FCs ranging from 26.5 to 58.9 and from 3.92 to
7.68, respectively. However, it is noteworthy that this change,
which is not considered a primary resistance mutation for EVG,
significantly compromised EVG susceptibility in the presence of
secondary resistance mutations (T97A plus V151I for one speci-
men and L74M for another).
tibilities. Comparisons of RAL and EVG FCs in the presence or
using nonparametric tests. Only samples without primary INI re-
sistance mutations were considered for this purpose, and there-
TABLE 1 Main characteristics of the study population
No. of samples examined
No. of samples from INI-naïve patients (%)
No. of samples from patients with RAL failures (%)
Median plasma HIV RNA level (log copies/ml) (IQR)
Median CD4 count (cells/mm3) (IQR)
% of samples of HIV-1 subtype
Median time of RAL therapya(wk) (IQR)
aOnly for the 40 samples belonging to patients failing on raltegravir-containing
regimens. INI, integrase inhibitor; RAL, raltegravir.
Garrido et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
fore, the analysis was carried out with a subset of 50 specimens.
Changes at 5 positions were positively associated with increased
FCs for RAL compared to the wild type. These changes were as
follows: S24G/N, D25E, L74I, K215N, and Q221H/S. On the con-
wild type. Of note, the L74I and K215N mutations appeared to-
gether in the same specimen, the same way most of the S24G/N
and D25E mutations appeared together. All this information is
summarized in Table 4. With respect to EVG, the L45Q/V,
V72I, and R263K mutations were associated with an increased
FC, while the S119G/P/S/T, V126M/L, and Q216H mutations
seemed to enhance EVG susceptibility in comparison with the
Cross-resistance between RAL and EVG. Bivariate associa-
tions between FCs for RAL and EVG expressed as log values re-
sulted in a significant positive correlation (R2? 0.8; P ? 0.001),
supporting that there was a high degree of cross-resistance be-
tween both drugs. However, reduced susceptibility to EVG was
more common than reduced susceptibility to RAL, as 27/61
(44.3%) samples displayed EVG resistance, in comparison with
only 15/61 (24.6%) samples showing reduced susceptibility to
RAL (P ? 0.002).
Resistance to both RAL and EVG has been described to be associ-
ated mainly with changes at integrase positions 148 and 155. Our
conferred high FCs to both drugs. The Y143R mutation leads to
the third RAL resistance pattern, and again, its impact on RAL
susceptibility was confirmed in our study, showing high FC val-
showing an FC of 4 to 8 when the Y143R mutation was present
along with other secondary changes. This is somewhat unex-
pected, since previous reports claimed that the Y143R mutation
did not influence EVG susceptibility (5, 14, 18). Altogether, our
results suggest that EVG activity is compromised in the presence
of any of the RAL resistance patterns, including the Y143R muta-
tion. Therefore, the sequential use of these drugs is not advisable
upon failure with either of them.
A few samples in our study displayed small, but potentially
clinically significant, reductions in susceptibility to integrase in-
of 2.0 and 1.9 for RAL and EVG, respectively. The reduced sensi-
resistance mutations. One specimen from a subject failing on a
RAL-containing regimen did not show any known INI resistance
This sample harbored only the R263K mutation as being poten-
the R263K mutation was previously shown to be selected in vitro
by EVG, conferring an FC of 5.2 to the drug (10). In the present
study, this mutation also led to a slight but significant decrease in
despite lacking primary INI resistance mutations. However, the
L74I mutation had been selected in this patient failing on a RAL-
resistance mutation generally selected along with the N155H mu-
tation in patients failing on RAL-containing regimens (8, 15), we
suggest that the L74I mutation might equally compromise RAL
Overall, the testing of samples collected from patients with
RAL failures in the absence of primary INI resistance mutations
allowed the recognition of certain minor changes that might im-
case for the S24G/N, D25E, L74I, K215N, and Q221H/S changes
for RAL and the L45Q/V, V72I, and R263K changes for EVG.
Additional resistance studies are being performed by Virco to
clear up the phenotypic effect of each of these changes. However,
their clinical impact should be further investigated with larger
groups of patients experiencing INI failures.
Most patients displaying resistance to RAL in our study also
exhibited a reduced EVG susceptibility. The extent of cross-resis-
tance was high for all different resistance patterns and may have
sequential use of RAL and EVG. Moreover, it was intriguing that
EVG showed increased FC values more often than RAL, suggest-
ing that integrase changes had a greater influence on EVG. Hypo-
thetically, our findings could be explained by the faster time of
dissociation from the viral integrase for EVG than for RAL, with
half-lives of 2.4 and 9 h, respectively (9).
The variability at the viral integrase has been shown to be
TABLE 2 Genotypic and phenotypic results for patients naïve to HIV integrase inhibitorsa
IDARV regimen Subtype
ABC, AZT, TDF, DRV/r
FTC, ddI, ATV
Q44K, I72V, T112IT, I113V, S119P, T122IT, E152G, T206S, T218S
D6E, E11D, V32I, V37I, L101I, T125A, I135V, V201I, S283G
E11D, E13D, V31I, V32I, S39C, M50I, I60V, L101I, I113V, T122I, T124N,
V32I, D41N, I72V, L101I, T122I, T124A, V201I
D6E, E11D, L28I, K34R, V37I, V201I, L234IL, D253DE, A265AV
AZT, 3TC, NVP
TDF, AZT, 3TC, ABC,
AZT, 3TC, fAPV/r
T97A E11D, S24G, D25E, L45V, M50I, L101I, T125A, F181L, T206S, D256E, S283G,
K14R, L101I, T112I, T124A, T125A, G134N, K136T, D167E, V201I, T206S,
Q221H, D232E, L234I, S255N, D256E, S283G
M50I, I113V, T124A, F181L, V201I, N222H, D256E
E11D, S24G, D25E, S39C, M50IM, I72V, L101I, V201I, S283GS
N_7FTC, TDF, fAPV/rG1.90
FTC, TDF, ATV/r
FTC, TDF, LPV/r
aART, antiretroviral therapy; FC, fold change; RAL, raltegravir; EVG, elvitegravir; ABC, abacavir; 3TC, lamivudine; FTC, emtricitabine; TDF, tenofovir; fAPV, fosamprenavir; ATV,
atazanavir; LPV, lopinavir; /r, ritonavir. Values in boldface indicate fold change above cutoff.
Elvitegravir and Raltegravir Cross-Resistance
June 2012 Volume 56 Number 6aac.asm.org 2875
TABLE 3 Genotypic and phenotypic results for patients failing on raltegravira
ETR, DRV/r, RAL
D6T, K7E, E13D, V31I, V79I, L101I, V201I
AZT, 3TC, ATV, RAL
E11D, V32I, S39C, L74I, V201I, I208L, K215N
MVC, ATV, RAL
M50I, I72V, S119P, T124A, V201I, I220L
3TC, MVC, RAL
E10D, V31I, I72V, L101I, S119G, T122I, T125A,
K156N, V201I, T206S, Q216H, Q221R, D256E
FTC, TDF, ETR, DRV/r, RAL
E11D, S24N, V31I, T112IM, T206S, I208L, D253DE
FTC, TDF, RAL
V31I, D41N, L101I, V201I, T206S, I268L, R269K
FTC, TDF, RAL
D6E, K14R, S17NS, V31I, L101I, K111EK, I135V,
G163Q, G193E, V201I, D256E
TDF, MVC, RAL
K14R, S24G, D25E, L45I, I113V, T124N, T125A,
K160S, V165IV, I220L, Y227F, L234V, D256E
S17N, L101I, T112I, V165I, H171L, T206S, D256E
3TC, ABC, RAL
E11D, K14R, K34R, D41N, P90S, L101I, K111KQ,
S119RS, K156N, I208L, D256E
AZT, 3TC, ETR, DRV/r, T20, RAL
K7R, S17N, R20K, L101I, T112V, S119P, T124A,
I135V, I220L, D232DN, D253DN
d4T, TDF, ETR, DRV/r, RAL
S17N, V31I, M50T, I72V, V75IV, T124A, K156N,
T206S, I220IV, D232DN, D256E
FTC, TDF, RAL
K7R, V31I, L101I, T112I, T125V, K219N, N222K
FTC, TDF, RAL
S17N, E96N, L101I, S119P, T122I, T124A, K156N,
V201I, T206S, I208L, Y227F, D253Y, D256E
FTC, TDF, ETR, RAL
D6E, L28I, V37I, S39C, I84IM, L101I, T124A,
AZT, 3TC, TDF, T20, DRV/r, RAL
K7KR, S17N, L101I, K111T, S119T, T124NS,
I208IL, I220L, Q221S, D253E, D256E
FTC, TDF, DRV/r, RAL
E10D, V32I, S39C, L101I, T218S, D253E
D6E, S17N, V32I, L45V, T124N, I203M, D253E
FTC, TDF, ETR, RAL
K7R, S17N, V31I, S39C, L101I, G163E, K173R,
3TC, LPV/r, RAL
V31I, L45Q, L101I, T206S, N254Q
FTC, TDF, RAL
K7R, K14R, L101I, T112I
aFC, fold change; RAL, raltegravir; EVG, elvitegravir; ?, undetectable RAL plasma levels; ?, detectable RAL plasma levels; DRV, darunavir; ETV, etravirine; MVC, maraviroc; ABC, abacavir; 3TC, lamivudine; FTC, emtricitabine; TDF,
tenofovir; fAPV, fosamprenavir, ATV, atazanavir; LPV, lopinavir; d4T, stavudine; T20, enfuvirtide; NA, not applicable. Values in boldface indicate fold change above cutoff.
Garrido et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
greater in antiretroviral-experienced than in drug-naïve patients
(1, 7, 21). All our patients, regardless of RAL exposure, were anti-
retroviral experienced, which might have led to a higher rate of
polymorphisms, providing an explanation of the relatively high
proportion of subjects with slight reductions in RAL and espe-
cially EVG susceptibilities in INI-naïve patients.
In summary, our results for the testing of phenotypic and ge-
notypic samples from patients with RAL failures and controls
support that susceptibility to RAL and EVG is impaired mainly
by the Q148H/R, N155H, and/or Y143R resistance mutation.
Slight reductions in susceptibility to any of these drugs can
occur in the presence of other integrase changes. The wide
extent of cross-resistance between RAL and EVG should pre-
clude their sequential use.
Members of the Spanish Integrase Resistance (SINRES) Group are as fol-
lows: J. Pedreira (Hospital Juan Canalejo, La Coruña, Spain); J. A. Iribar-
ren (Hospital de Donostia, San Sebastián, Spain); J. Solá (Hospital de
Navarra, Pamplona, Spain); M. J. Téllez and V. Estrada (Hospital Clínico
San Carlos, Madrid, Spain); S. García and J. R. Arribas (Hospital La Paz,
Madrid, Spain); L. Santos (Hospital de la Princesa, Madrid, Spain); P.
Miralles (Hospital Gregorio Marañón, Madrid, Spain); J. E. Losa (Hospi-
tal Universitario Fundación de Alcorcón, Madrid, Spain); M. Cervero
(Hospital Severo Ochoa, Madrid, Spain); J. Sanz (Príncipe de Asturias,
Madrid, Spain); C. Barros (Hospital de Móstoles, Madrid, Spain); M. de
Górgolas (Fundación Jiménez Díaz, Madrid, Spain); E. Ribera and E. Ca-
(Hospital Son Dureta, Palma de Mallorca, Spain); C. Gómez (Complejo
Hospitalario de Toledo); A. Chocarro (Virgen de la Concha, Zamora,
tal Ciudad de Jaén); A. Collado and C Gálvez (Hospital Torrecardenas,
Almeria, Spain); A. B. Lozano and J. M. Fernández (Hospital Poniente,
sitario Virgen de las Nieves, Granada, Spain); F. García, V. Guillot, and J.
Hernández-Quero (Hospital Universitario San Cecilio Granada); J. A.
Mendoza, and V. Soriano (Hospital Carlos III, Madrid, Spain).
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TABLE 4 Phenotypic impact of minor changes in the HIV integrase gene in the absence of primary resistance mutations on susceptibility to
raltegravir and elvitegravira
Median FC (IQR) (no. of specimens)
1.05 (0.70–1.30) (46)
1.10 (0.70–1.30) (47)
1.10 (0.75–1.3) (49)
1.05 (0.7–1.3) (46)
1.10 (0.8–1.3) (47)
1.65 (1.27–2.25) (4)
1.80 (1.20–2.40) (3)
1.4 (1.3–1.8) (4)
0.7 (0.6–0.8) (3)
1.19 (0.9–2.08) (47)
1.14 (0.76–1.44) (18)
1.2 (0.92–2.22) (49)
1.48 (1.09–2.45) (33)
1.3 (0.98–2.33) (48)
1.3 (0.98–2.33) (48)
2.63 (2.43–2.80) (3)
1.48 (1–2.42) (32)
1 (0.70–1.39) (17)
0.76 (0.70–0.76) (2)
0.62 (0.48–0.76) (2)
aFC, fold change; RAL, raltegravir; EVG, elvitegravir; WT, wild type. Values in boldface indicate fold change above cutoff.
Elvitegravir and Raltegravir Cross-Resistance
June 2012 Volume 56 Number 6 aac.asm.org 2877
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