Evolution of genotypic resistance to enfuvirtide in HIV-1 isolates from different group M subtypes.
ABSTRACT Enfuvirtide is active against isolates from different HIV-1 subtypes. In vitro and in vivo studies reveal that resistance mutations are primarily found within the region spanning amino acid 36-45 of gp41. However, most studies include only subtype B strains, while it is known that especially the env region is very divergent among subtypes.
To analyze the gp41 HR1 genetic evolution during failure of enfuvirtide-containing salvage regimens in 19 HIV-1 patients infected with strains from different group M subtypes.
The gp41 sequence was determined at baseline and upon failure in 19 patients. For a subset of 7 patients, samples were available after discontinuation of enfuvirtide.
Our results confirmed the conserved nature of the HR1 region. Escape mutants during chronic treatment with enfuvirtide were mainly observed within region 36-45. One novel mutation was identified, i.e. S42G in a subtype A1 strain.
Different subtypes escape enfuvirtide selective pressure through similar mutational patterns, however a new S42G variant was observed. The in vivo selection of S42G suggests that it might play a role in enfuvirtide resistance. Therefore, it could be considered as a candidate mutation to be included within drug resistance interpretation systems.
- SourceAvailable from: Lucia Palmisano[Show abstract] [Hide abstract]
ABSTRACT: The European HIV Drug Resistance Guidelines Panel, established to make recommendations to clinicians and virologists, felt that sufficient new information has become available to warrant an update of its recommendations, explained in both pocket guidelines and this full paper. The Panel makes the following recommendations concerning the indications for resistance testing: for HIV-1 (i) test earliest sample for protease and reverse transcriptase drug resistance in drug-naive patients with acute or chronic infection; (ii) test protease and reverse transcriptase drug resistance at virologic failure, and other drug targets (integrase and envelope) if such drugs were part of the failing regimen; (iii) consider testing for CCR5 tropism at virologic failure or when a change of therapy has to be made in absence of detectable viral load, and in the latter case test DNA or last detectable plasma RNA; (iv) consider testing earliest detectable plasma RNA when a successful nonnucleoside reverse transcriptase inhibitor-containing therapy was inappropriately interrupted; (v) genotype source patient when postexposure prophylaxis is considered; for HIV-2, (vi) consider resistance testing where treatment change is needed after treatment failure. The Panel recommends genotyping in most situations, using updated and clinically evaluated interpretation systems. It is mandatory that laboratories performing HIV resistance tests take part regularly in external quality assurance programs, and that they consider storing samples in situations where resistance testing cannot be performed as recommended. Similarly, it is necessary that HIV clinicians and virologists take part in continuous education and discuss problematic clinical cases. Indeed, resistance test results should be used in the context of all other clinically relevant information for predicting therapy response.AIDS reviews 01/2011; 13(2):77-108. · 4.02 Impact Factor
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ABSTRACT: Mutations that are selected at low frequency and/or reside outside the enfuvirtide target region, amino acid 36-45 of gp41, might still be important determinants for drug resistance. This study aimed to investigate the phenotypic impact against enfuvirtide and sifuvirtide of uncharacterized gp41 mutations 42G, 43T and 50V, selected in patients failing enfuvirtide-containing regimens. As single mutations, neither 42G, 43T nor 50V conferred resistance to enfuvirtide. However, 50V increased slightly resistance levels for 36D, 38M, 43D or 43T as did 43T for 38M. All mutants displayed a reduced replication capacity, except 42S, 50V and 36D+/-50V. None of the mutants displayed resistance to the next-generation fusion inhibitor sifuvirtide. This study highlights the necessity to confirm the in vitro effect of infrequently selected mutations as 42G was not associated with enfuvirtide resistance whereas 43T and 50V should be considered as secondary enfuvirtide resistance mutations.Antiviral research 03/2010; 86(3):253-60. · 3.61 Impact Factor
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ABSTRACT: On the basis of mutagenesis, biochemical, and structural studies, heptad repeat 1 of HIV gp41 (HR1) has been shown to play numerous critical roles in HIV entry, including interacting with gp120 in prefusion states and interacting with gp41 heptad repeat 2 (HR2) in the fusion state. Moreover, HR1 is the site of therapeutic intervention by enfuviritide, a peptide analogue of HR2. In this study, the functional importance of each amino acid residue in gp41 HR1 has been systematically examined by alanine scanning mutagenesis, with subsequent characterization of the mutagenic effects on folding (as measured by incorporation into virions), association with gp120, and membrane fusion. The mutational effects on entry can be grouped into three classes: (1) wild type (defined as >40% of wild-type entry), (2) impaired (defined as 5-40% of wild-type entry), and (3) nonfunctional (defined as <5% of wild-type entry). Interestingly, the majority of HR1 mutations (77%) exhibit impaired or nonfunctional entry. Surprisingly, effects of mutations on folding, association, or fusion are not correlated to heptad position; however, folding defects are most often found in the N-terminal region of HR1. Moreover, disruption of the gp41-gp120 interaction is correlated to the C-terminal region of HR1, suggesting that this region interacts most closely with gp120. In summary, the sensitivity of gp41 HR1 to alanine substitutions suggests that even subtle changes in the local environment may severely affect envelope function, thereby strengthening the notion that HR1 is an attractive site for therapeutic intervention.Biochemistry 06/2010; 49(24):5057-65. · 3.38 Impact Factor
Journal of Clinical Virology 44 (2009) 325–328
Contents lists available at ScienceDirect
Journal of Clinical Virology
journal homepage: www.elsevier.com/locate/jcv
Evolution of genotypic resistance to enfuvirtide in HIV-1 isolates from
different group M subtypes
Kris Covensa,∗, Kabamba Kabeyab, Yoeri Schrootena,c, Nathalie Dekeersmaekera,
Eric Van Wijngaerdend, Anne-Mieke Vandammea, Stéphane De Witb,
Kristel Van Laethema,c
aRega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
bAIDS Reference Centre, Division of Infectious Diseases, CHU Saint-Pierre, Brussels, Belgium
cAIDS Reference Laboratory, University Hospitals Leuven, Leuven, Belgium
dInternal Medicine, University Hospitals Leuven, Leuven, Belgium
a r t i c l ei n f o
Received 29 October 2008
Received in revised form 21 January 2009
Accepted 23 January 2009
a b s t r a c t
However, most studies include only subtype B strains, while it is known that especially the env region is
very divergent among subtypes.
Objectives: To analyze the gp41 HR1 genetic evolution during failure of enfuvirtide-containing salvage
regimens in 19 HIV-1 patients infected with strains from different group M subtypes.
Study design: The gp41 sequence was determined at baseline and upon failure in 19 patients. For a subset
of 7 patients, samples were available after discontinuation of enfuvirtide.
Results: Our results confirmed the conserved nature of the HR1 region. Escape mutants during chronic
treatment with enfuvirtide were mainly observed within region 36–45. One novel mutation was identi-
fied, i.e. S42G in a subtype A1 strain.
however a new S42G variant was observed. The in vivo selection of S42G suggests that it might play a role
in enfuvirtide resistance. Therefore, it could be considered as a candidate mutation to be included within
drug resistance interpretation systems.
© 2009 Elsevier B.V. All rights reserved.
Enfuvirtide is a peptide of 36 amino acids (AA) that mimics a
part of the second heptad repeat region (HR2) of HIV-1 gp41 that
is involved in membrane fusion between virus and target cell.1The
peptide sequence was based on the gp41 sequence of the subtype B
LAI strain.2Upon binding with the first heptad repeat region (HR1),
it prevents fusion of viral and cellular membrane and thus inhibits
the viral replicative cycle.
HR1 is highly conserved among the different HIV-1 group M
higher degree of genetic variability.3,4However, the natural genetic
∗Corresponding author at: Rega Institute for Medical Research, Microbiology and
Immunology, Clinical and Epidemiological Virology, Minderbroedersstraat 10, 3000
Leuven, Belgium. Tel.: +32 16 332177; fax: +32 16 332131.
E-mail address: firstname.lastname@example.org (K. Covens).
variability within HR1 and HR2 does not appear to have an impact
on the virological response to enfuvirtide therapy in enfuvirtide-
naïve patients.6There is even evidence of activity against HIV-1
group O, both in vitro and in vivo, albeit only one patient had been
subject of investigation.7,8
Most mutations associated with enfuvirtide resistance have
HR1 of gp41.5These mutations have been implemented in various
on enfuvirtide resistance have included mainly subtype B strains.
The envelope region displays 30–35% divergence between differ-
ent subtypes as opposed to 10–15% for pol.9Although most studies
including protease and reverse transcriptase inhibitors have shown
similar virological responses and mutational patterns in patients
infected with non-B strains in comparison to subtype B strains,
there are nevertheless some reports that reveal subtype-specific
resistance mutations.10–14Therefore, the goal of this study was
to enlarge knowledge on enfuvirtide resistance in HIV-1 strains
from different subtypes and to search for yet unreported mutations
selected by enfuvirtide.
1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
K. Covens et al. / Journal of Clinical Virology 44 (2009) 325–328
Fig. 1. Sequence analysis of the HIV-1 HR1 domain (gp41 position 29–82) from patients failing enfuvirtide-containing therapy. The amino acid sequence 36–45,
previously associated with enfuvirtide resistance, is displayed in bold. Samples are annotated with their isolation date, as weeks following enfuvirtide initiation (+)
or discontinuation (−). The maximum period on/off enfuvirtide pressure before discontinuation/recontinuation is displayed in bold. For some patients at certain
K. Covens et al. / Journal of Clinical Virology 44 (2009) 325–328
2. Patients and methods
Samples from 19 highly therapy-experienced patients attend-
ing CHU St. Pierre Hospital and University Hospitals Leuven
and virologically failing enfuvirtide-containing regimens were
included. Virological failure was defined as a viral load above
the detection limit of the AMPLICOR HIV-1 monitor test, i.e.
400RNAcopies/ml or 50copies/ml. The median duration of enfu-
virtide treatment was 96 weeks (range 24–289). Extraction,
reverse transcription, amplification and sequencing were per-
formed as described previously.15Alternatively, one-step RT-PCR
was carried out using SuperScript III One-Step RT-PCR System
with Platinum Taq High Fidelity (Invitrogen, Merelbeke, Belgium)
with primers EnvA (NL4.3 Genbank accession number AF324493
NT 5953–5980 5?-GGCTTAGGCATCTCCTATGGCAGGAAGAA-3?) and
KVL009 (5?-GCCAATCAGGGAAGWAGCCTTGTGT-3?) and the cycling
conditions: 55◦C for 30min, 94◦C for 2min followed by 40
cycles of 15s at 94◦C, 30s at 55◦C, 3min 30s at 68◦C and a
final extension of 5min at 68◦C. 1?l of outer PCR was used
as template for an inner PCR carried out with Platinum Taq
DNA Polymerase High Fidelity (Invitrogen) with primers EnvB
(NT 6198–6224 5?-AGAAAGAGCAGAAGACAGTGGCAATGA-3?) and
EnvM (NT 9058–9086 5?-TAGCCCTTCCAGTCCCCCCTTTTCTTTTA-3?).
Cycling conditions were 2min at 94◦C, followed by 30 cycles of
15s at 94◦C, 30s at 55◦C, 2min 45s at 68◦C and a final elonga-
tion of 5min at 68◦C. The envelope sequences were subtyped by
by a manual phylogenetic analysis including 60 reference envelope
sequences (two sequences for each pure subtype and 33 CRF) from
the Los Alamos HIV database (http://www.hiv.lanl.gov).
3. Results and discussion
Nineteen patients were studied, 7 infected with a subtype B, 2
with a subtype A, 1 with a subtype C, 3 with a subtype D, 3 with
a subtype G, 1 with a CRF02 AG and 2 with a unique recombinant
form (URF) (Fig. 1). The first URF was a recombination of subtype A
and J, whereas the second one consisted of subtype A and G.
An alignment of gp41 AA positions 29–82 of the available
samples with respective consensus sequences per subtype or
recombinant form is shown in Fig. 1. Our results confirm the
conserved nature of the 36–45 stretch within group M.3,4Polymor-
phisms at position 42 were frequently occurring. The consensus is
S42 among all genetic forms within this study, except for subtype B
N42 at baseline. Additionally, one other subtype B strain displayed
at baseline a mixture of IM at position 37 that was not observed
anymore in subsequent samples.
All but three patients displayed genetic evolution within the
region 36–45 upon failure of the enfuvirtide-containing regimens.
Two of these three patients failed with low viral loads throughout
administration of the enfuvirtide-regimen (B-1 between 500 and
the sampling for the third patient (B-5) occurred 12 weeks after
nadir viral load and reversion to wild-type might have occurred
due to adherence issues.
was altered in 9 patients, i.e. A in 6, E in 1 and M in 3. The second
G (1), I (1), N (1) or T (3), and N43 that also evolved in 7 patients,
but to D (3), K (2), S (1) or T (1). Other altered AA in the 36–45
stretch were G36D (1), Q40H (5) and L45M (3). The majority of the
observed mutations have been reported before, however, 1 novel
mutation was identified, i.e. S42G in a subtype A1 (A-1).
In 10 patients, combinations of mutations occured, some
of which were only present transiently. The mutational pat-
tern changed from V38A to N42T+N43S in 1 patient (B-2).
As observed within their population sequences (B-6, B-7), two
other patients displayed the mixtures V38VA+Q40QH+L45LM
that subsequently lost the 38A variant and evolved to the pure
combination Q40H+L45M. This could indicate that the benefit
in terms of reduced susceptibility to enfuvirtide and impact on
viral replication capacity of the combination Q40H+L45M might
be higher than V38A+Q40H, V38A+L45M or the combination of
all 3 mutations. Samples from 4 patients showed combinations of
mutations at positions 36, 38 and 43, for which a highly exclu-
sive relationship has been reported before,17i.e. V38M+N43K
in patient B-4, G36GD+V38VM in D-2, V38M+N43T in G-2 and
V38VAE+S42ND+N43ND in G-3. In agreement with the reported
exclusive relationships, the mixed population G36GD+V38VM
changed to N42T in patient D-2, whereas in patient G-3 an evo-
lution towards V38A+S42ND was observed. However, in the other
two patients no mixtures were observed at codons 38 and 43, indi-
cating that at least the majority of variants contain both mutations.
This might suggest that these particular AA changes are compatible
within the respective patient background sequence.
For 7 patients of whom 6 displayed resistance associated muta-
tions on treatment, samples after discontinutation of enfuvirtide
were available for analysis. In all but one of them, reversion to
wild-type or mixtures of wild-type and mutant was detected. Due
to adherence problems, reversion occurred already in one patient
while on enfuvirtide therapy (C-1). In patient B-4, the one extra
mutation V38M detected after 14 weeks of enfuvirtide discontin-
uation is most likely developed during the additional 47 weeks
of enfuvirtide treatment administered after the last on treatment
sample implemented within our study.
within group M and that escape mutants surviving treatment with
Thus, despite our restriction to the investigation of only the HR1
genetic evolution during failure of enfuvirtide-containing therapy
within a limited number of non-B subtypes, these results might
the selection of similar major mutations.
Nevertheless, one novel mutation, S42G, has been identified
within a non-B subtype. If phenotypic testing would confirm its
impact on enfuvirtide susceptibility, S42G could be a candidate
mutation for inclusion within drug resistance interpretation sys-
Conflict of interest statement
All authors declare to have no conflicts of interest.
We are grateful to Ana Carolina Palma for excellent technical
assistance. This work was supported by the AIDS Reference Labo-
time points, samples were not available anymore for analysis. Genetic forms were determined by submitting the sequences to the REGA HIV-1 subtyping tool16
(http://jose.med.kuleuven.be/subtypetool/html/index.html). Only the differences between the ancestor (anc) group M sequence and the consensus (con) sequence for each
subtype, circulating recombinant form (CRF), unique recombinant form (URF), as obtained from HIV databases (http://www.hiv.lanl.gov), or the respective patient derived
sequences are displayed in one letter symbols. Mixtures of wild-type and mutant are displayed in lower case. Mixtures of multiple mutants are displayed in subscript and
K. Covens et al. / Journal of Clinical Virology 44 (2009) 325–328
ratory of Leuven that receives support from the Belgian Ministry of
Social Affairs through a fund within the Health Insurance System,
by the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (grant
G.0266.04) and by the Centers of Excellence of the Katholieke Uni-
versiteit Leuven (krediet EF/05/015). Kris Covens was funded by a
PhD grant of the Institute for the Promotion of Innovation through
Sciences and Technology in Flanders (IWT).
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