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: email@example.com (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).
1. Kilgore NR, Salzwedel K, Reddick M, Allaway GP, Wild CT. Direct evidence
that C-peptide inhibitors of human immunodeficiency virus type 1 entry
bind to the gp41 N-helical domain in receptor-activated viral envelope. J Virol
2. Wild CT, Shugars DC, Greenwell TK, McDanal CB, Matthews TJ. Peptides cor-
responding to a predictive alpha-helical domain of human immunodeficiency
virus type 1 gp41 are potent inhibitors of virus infection. Proc Natl Acad Sci USA
3. Aghokeng AF, Ewane L, Awazi B, Nanfack A, Delaporte E, Zekeng L, et al. Enfu-
virtide binding domain is highly conserved in non-B HIV type 1 strains from
Cameroon, West Central Africa. AIDS Res Hum Retroviruses 2005;21:430–3.
4. Razzolini F, Vicenti I, Saladini F, Micheli V, Romano L, Cargnel A, et al. Natural
variability in the HR-1 and HR-2 domains of HIV type 1 gp41 from different
clades circulating in Italy. AIDS Res Hum Retroviruses 2007;23:558–63.
5. Sista PR, Melby T, Davison D, Jin L, Mosier S, Mink M, et al. Characterization of
determinants of genotypic and phenotypic resistance to enfuvirtide in baseline
and on-treatment HIV-1 isolates. AIDS 2004;18:1787–94.
6. Melby T, Sista P, DeMasi R, Kirkland T, Roberts N, Salgo M, et al. Characteriza-
tion of envelope glycoprotein gp41 genotype and phenotypic susceptibility to
enfuvirtide at baseline and on treatment in the phase III clinical trials TORO-1
and TORO-2. AIDS Res Hum Retroviruses 2006;22:375–85.
7. Chinnadurai R, Münch J, Dittmar MT, Kirchhoff F. Inhibition of HIV-1 group M
and O isolates by fusion inhibitors. AIDS 2005;19:1919–22.
8. Poveda E, Barreiro P, Rodés B, Soriano V. Enfuvirtide is active against HIV type
1 group O. AIDS Res Hum Retroviruses 2005;21:583–5.
9. Renjifo B, Essex M. HIV-1 subtypes and recombinants. In: AIDS in Africa. 2nd ed.
New York: Kluwer Academic-Plenum Publishers; 2002. p. 138–57.
10. Abecasis AB, Deforche K, Snoeck J, Bacheler LT, McKenna P, Carvalho AP, et al.
Protease mutation M89I/V is linked to therapy failure in patients infected with
the HIV-1 non-B subtypes C, F or G. AIDS 2005;19:1799–806.
11. Abecasis AB, Deforche K, Bacheler LT, McKenna P, Carvalho AP, Gomes P, et al.
Investigation of baseline susceptibility to protease inhibitors in HIV-1 subtypes
C, F, G and CRF02 AG. Antivir Ther 2006;11:581–9.
12. Brenner B, Turner D, Oliveira M, Moisi D, Detorio M, Carobene M, et al. A V106M
mutation in HIV-1 clade C viruses exposed to efavirenz confers cross-resistance
to non-nucleoside reverse transcriptase inhibitors. AIDS 2003;3:F1–5.
13. Brenner BG, Oliveira M, Doualla-Bell F, Moisi DD, Ntemgwa M, Frankel F, et al.
HIV-1 subtype C viruses rapidly develop K65R resistance to tenofovir in cell
culture. AIDS 2006;20:F9–13.
14. Deforche K, Silander T, Camacho R, Grossman Z, Soares MA, Van Laethem K, et
al. Analysis of HIV-1 pol sequences using Bayesian Networks: implications for
drug resistance. Bioinformatics 2006;22:2975–9.
15. Van Laethem K, Schrooten Y, Lemey P, Van Wijngaerden E, De Wit S, Van
Ranst M, et al. A genotypic resistance assay for the detection of drug resistance
in the human immunodeficiency virus type 1 envelope gene. J Virol Methods
16. de Oliveira T, Deforche K, Cassol S, Salminen M, Paraskevis D, Seebregts C, et al.
An automated genotyping system for analysis of HIV-1 and other microbial
sequences. Bioinformatics 2005;21:3797–800 (http://jose.med.kuleuven.be/
evolution of gp41 reveals a highly exclusive relationship between codons 36, 38
and 43 in gp41 under long-term enfuvirtide-containing salvage regimen. AIDS