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Previous Article | Next Article 
Journal of Virology, January 1999, p. 850-854, Vol. 73, No. 1
Service de Médecine Interne,
Received 27 July 1998/Accepted 15 October 1998
Human immunodeficiency virus type 1 (HIV-1) variants that have
developed protease (PR) inhibitor resistance most often display cross-resistance to several molecules within this class of
antiretroviral agents. The clinical benefit of the switch to a second
PR inhibitor in the presence of such resistant viruses may be
questionable. We have examined the evolution of HIV-1 PR genotypes and
phenotypes in individuals having failed sequential treatment with two
distinct PR inhibitors: saquinavir (SQV) followed by indinavir (IDV).
In viruses where typical SQV resistance mutations were detected before the change to IDV, the corresponding mutations were maintained under
IDV, while few additional mutations emerged. In viruses where no SQV
resistance mutations were detected before the switch to IDV, typical
SQV resistance profiles emerged following the introduction of IDV. We
conclude that following suboptimal exposure to a first PR inhibitor,
the introduction of a second molecule of this class can lead to rapid
selection of cross-resistant virus variants that may not be detectable
by current genotyping methods at the time of the inhibitor switch.
Viruses committed to resistance to the first inhibitor appear to bear
the "imprint" of this initial selection and can further adapt to
the selective pressure exerted by the second inhibitor following a
pathway that preserves most of the initially selected mutations.
Inhibitors of the human
immunodeficiency virus type 1 (HIV-1) protease (PR) are among the most
active antiretroviral compounds used in the therapy of HIV-1 infection
(8). These agents impair the maturation and the infectivity
of viral particles and lead to a rapid blocking of virus replication
(1, 7, 11, 16, 21, 30). Combinations of antiretroviral
agents that include a PR inhibitor can drive the levels of HIV-1 RNA in
the plasma below the limit detectable by current quantitation
techniques and lead to long-term clinical and immunological
improvements (2, 12, 19). Nevertheless, suboptimal therapies
that fail to achieve a complete and sustained inhibition of virus
replication usually lead to the selection of viral variants with
decreased susceptibility to the corresponding agents (6, 10, 15, 18, 23). Mutations in the viral PR that can confer resistance to
PR inhibitors have been thoroughly described both in vitro and in vivo
(5, 20, 22, 23, 25, 26, 28). Selection for HIV-1 resistance
to PR inhibitors is a complex process that can recruit mutations
located in different sites of the PR. Although they may differ from one
PR inhibitor to another, there seems to be considerable overlap in the
mutational pathways leading to resistance to most currently used PR
inhibitors (22). Resistance to saquinavir (SQV) is
characterized by the selection of mutations L90M and/or G48V (15,
28, 31, 32). Resistance to indinavir (IDV) in most cases involves
early selection of mutation V82A (5, 6). However, most
initially selected resistance mutations are insufficient to induce
high-level resistance, which requires the addition of secondary
mutations that are often located outside of the active site of the PR,
such as L10I, M36I, M46I, L63P, or A71V. Such secondary mutations,
which increase the level of resistance and often correct the loss of
fitness that can characterize resistant viruses, are observed during
selection of most PR inhibitors (3, 14, 17, 22, 24, 29). As
a consequence, resistant variants most often display significant
cross-resistance to several inhibitors of the same class (6, 13,
32). In spite of accumulating in vitro data on cross-resistance
between PR inhibitors, the clinical significance of cross-resistance is
still unclear. In particular, the impact of resistance to one PR
inhibitor on the virological response to a second molecule of the same
class has not been fully evaluated. We have examined the evolution of
HIV-1 genotypes and phenotypes in patients who have received SQV
followed by IDV. Since virological failure of SQV has been reported to
be often accompanied by the persistent replication of viruses that do
not bear detectable resistance mutations in the PR (15, 28),
we also wished to determine whether such apparently sensitive viruses would retain full in vivo responsiveness to subsequent therapy by
another PR inhibitor.
Eleven patients who had failed sequential PR inhibitor therapy with SQV
followed by IDV were retrospectively selected out of a population of 54 patients who had received SQV (hard-gel capsule formulation, 600 mg
three times per day) as part of a clinical trial or of an expanded use
program at the Antoine Béclère Hospital. Because the aim of
the study was an analysis of the evolution of viruses under sequential
selective pressure by distinct PR inhibitors rather than an evaluation
of the response to such a regimen in treated patients, the criteria for
treatment failure and patient selection in this study were arbitrarily
defined in order to include viruses that clearly escaped both PR
inhibitors. Therefore, treatment failure was defined as a reduction in
plasma viremia of less than 1 log10. Of the 54 selected
patients, 26 were considered to have failed SQV therapy. Twenty-two of
these 26 patients were switched to IDV (800 mg three times per day) with or without concurrent changes in other antiretroviral drugs. Of
the 22 patients who were switched to IDV, 11 were considered to have
failed IDV therapy and were subsequently studied. Four of these
patients (patients C, D, G, and J) (Table
1) had a transient response to IDV
characterized by a drop in plasma viremia in excess of 1 log10, but their viremia subsequently rebounded to within 1 log10 of viremia at the time of the switch. For all
subjects, HIV-1 PR sequences were amplified from plasma virus by nested reverse transcription (RT)-PCR using the primer pair ProA+
(5' GCT AAT TTT TTA GGG AAG ATC TG 3') and ProA The evolution of HIV-1 PR sequences in the 11 study patients before and
after the switch from SQV to IDV is presented in Table 1. According to
this evolution, we subdivided the 11 patients into the following three
groups: group I (patients A through E), in which significant genotypic
PR resistance profiles resulting from SQV treatment were present at
month 0 (M0), the time of the switch; group II (patients F
and G), in which relatively little evolution of PR had occurred before
M0; and group III (patients H through K), in which HIV-1 PR
amino acid sequences did not bear detectable resistance mutations at
M0. In group I, patients A, B, and C had received SQV for
more than 6 months before the earliest sequence was available. Several
months before the switch, all three patients displayed the
characteristic SQV-resistance mutation L90M, which is associated with
other amino acid changes. Comparable amino acid changes were observed
in patients D and E at M0. After the switch from SQV to
IDV, all five viruses retained the L90M mutation with the exception of
a relative and transient decrease of that mutation at M3 in
virus populations from patient C. In this group of viruses, a few
additional mutations emerged following the switch, involving codons 10, 46, 71, and 73. However, none of the five viruses required the addition
of a mutation at codon 82, most often associated with IDV resistance.
Therefore, in this group, mutations selected under SQV treatment
appeared to induce sufficient cross-resistance to IDV to allow
significant virus replication in the presence of IDV in vivo. In the
two viruses in group II, only one mutation had been selected during the
course of SQV treatment. Compared to standard HIV-1 subtype B
sequences, virus from patient F displayed amino acid changes at
positions 10, 20, and 36 which are often observed during PR resistance, but which, because the corresponding virus had not been exposed to SQV,
were more likely to be a natural polymorphism than the result of SQV
selection. In this virus, the SQV-resistance mutation G48V was present
as a minority at M0 and was maintained thereafter. Moreover, the V82A mutation was observed at M3, a frequent
association with the G48V mutation in viruses with high-level
resistance to SQV (32). In patient G, the only mutation
selected at M0 was A71V, which was followed by the addition
of several other resistance mutations, including V82A. In group III,
plasma virus sequences displayed no detectable resistance mutations at
the time of the switch to IDV. Careful examination of the sequence
chromatograms at M0 failed to reveal even minor species of
mutant genomes. Surprisingly, the resistance mutations that developed
in these viruses after the switch were very similar to those described
above for patients in group I, which had developed resistance mutations
under SQV treatment. In particular, under the selective pressure of
IDV, all four viruses in group III developed the L90M mutation, which is associated with remarkably similar combinations of mutations at
codons 10, 46, 71, and 73.
To confirm the results of the genotypic analysis reported above, HIV-1
SQV and IDV susceptibility phenotypes were examined sequentially in
nine of the patients. We used a rapid recombination method that allows
testing of recombinant virus carrying plasma-derived PR sequences
(27). Briefly, PR sequences were amplified from plasma by
RT-PCR using the same primers as for PR sequencing and were recombined
by cotransfection in HeLa cells with a linearized pNL4-3-derived
plasmid carrying a full deletion of PR coding sequences. The resulting
recombinant virus was tested for susceptibility to SQV and IDV on
HeLa-derived indicator P4 cells, as previously described
(33). Drug susceptibility was expressed as IC90
values (the drug concentration required to inhibit 90% of HIV
replicative events) using the median effect method (4). The
results of this phenotyping analysis are presented in Table
2. For patients E, G, H, I, and K, the
parental pre-SQV virus was used as the reference to calculate the
decrease in drug susceptibility. For patient J, in which the pretherapy
virus was not tested, we used the M0 virus, which was
obtained at the time of the switch and was free of detectable
resistance mutations, as the reference. For patients A, C, and D, in
which genotypic resistance had developed before the first tested plasma
sample was obtained, the reference was pNL4-3 virus. In all patients,
as shown in Table 2, measurable dual resistance to SQV and IDV
developed, with a gradual increase over time in viral resistance to
both inhibitors. With the notable exception of virus from patient H,
there was a general trend towards a higher resistance to SQV than to
IDV. In the four tested group I viruses (from patients A, C, D, and E),
which displayed clear SQV resistance genotypes at M0,
phenotypic testing revealed an increase in SQV resistance of 5.8-, 6.4-, 19.2-, and 8-fold, respectively. Viruses from patients A and E
displayed an increase in resistance to both SQV and IDV over time,
while virus from patient C, in which the L90M mutation was temporarily
reduced to a minority at M3, showed a corresponding
decrease of resistance to both inhibitors. Therefore, as noted from the
genotypic analysis of viruses of this group, the initial resistance to
SQV was in most instances not only maintained, but clearly reinforced
by the switch to IDV. In virus from patient G, in which a single A71T
mutation emerged at M0, followed by numerous other
resistance mutations, including L90M and V82A (a typical IDV resistance
mutation), a parallel increase in the level of resistance to both drugs
was measurable after M0. Finally, as expected from their
genotypes, viruses in group III had no significant phenotypic
resistance to either of the two drugs at M0. Following the
switch from SQV to IDV, these four viruses displayed a decrease of
their susceptibility to both drugs. In patient H, the M10
virus was clearly more resistant to IDV than to SQV. However, in the
three other patients (subjects I, J, and K), the increase in viral
resistance was significantly more prominent regarding SQV. Again, in
these cases, the phenotypic analysis confirmed the genotype data
showing that viruses that were selected following the switch from SQV
to IDV were in fact mainly SQV-resistant viruses.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Constrained Evolution of Human Immunodeficiency
Virus Type 1 Protease during Sequential Therapy with Two Distinct
Protease Inhibitors
ABSTRACT
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(5' GGC
AAA TAC TGG AGT ATT GTA TG 3') and the pair ProB+ (5' TTT
TTA GGG AAG ATC TGG CCT TC 3') and ProB (5' GGA GTA TTG
TAT GGA TTT TCA GG 3'). Population-based sequencing of the PR coding
region was performed by the dideoxy-termination method on automated ABI
sequencers using bulk second-round PCR products as templates and
primers PRO1 (sense, 5' CCC TCT CAG AAG CAG GAG 3') and PRO2
(antisense, 5' TGG GCC ATC CAT TCC TGG CTT 3'). Plasma virus loads were
determined with the Amplicor kit (Roche).
TABLE 1.
PR mutations in plasma-derived HIV-1 sequences from PR
inhibitor-treated subjects
TABLE 2.
IC90s of IDV and SQV
The results of this study reveal that viruses selected in vivo for SQV resistance display a significant cross-resistance to IDV. As a result of this cross-resistance, the switch to IDV maintained the SQV-selected mutations, while only a small number of additional mutations emerged as the result of IDV selective pressure. The corresponding viruses, bearing the "imprint" of SQV selection, were not required to radically modify their pattern of PR-resistance mutations. In particular, only a few of the viruses selected here needed a mutation of codon 82, most frequently observed during selection for IDV resistance. This can be explained by the fact that the cross-resistance to IDV resulting from the initial SQV selection is sufficient for in vivo resistance to IDV. It could also be explained by a possible relative incompatibility between mutation L90M and V82A in terms of viral replicative fitness. Indeed, it has been recently reported that in virus populations where mutations of codon 82 develop as a result of SQV selection, quasi-species bearing the L90M mutation are often gradually eliminated over time (9). Correspondingly, mutations of codon 82 are significantly more often observed in association with G48V, a mutation able to confer high-level resistance to SQV, than with L90M.
More interestingly, viruses with no detectable resistance mutations at the time of the introduction of IDV (group III viruses) rapidly developed a pattern of genotypic and phenotypic resistance predominantly to SQV, even after removal of that drug. In these cases, it can be assumed that minor virus variants bearing SQV-resistance mutations failed to be selected by SQV, probably due to an insufficient selective pressure provided by the poorly bioavailable formulation of SQV used in this study. Because of the insufficient amount of active drug and also presumably because of a slightly reduced viral fitness in mutant quasispecies compared to that of wild-type virus, these variants failed to significantly take over wild-type virus before the inhibitor switch. After the introduction of IDV, by virtue of the significant SQV-IDV cross-resistance measured here and of the better bioavailability of IDV, the mutant quasi-species were readily selected, again bearing the imprint of SQV selection. Later, these imprinted viruses behaved similarly to the SQV-selected viruses found in the other patients, withholding their mutation selection pathway.
It should be feared that such a scenario can be applied to many instances where PR inhibitors are used sequentially. Indeed, one can assume that in cases of insufficient virological response to a first PR inhibitor, even if resistant variants are not perceptible by current genotyping or phenotyping methods and provided that they display sufficient cross-resistance to the second PR inhibitor, these variants will be selected following the switch and will gradually emerge, leading to therapeutic failure of the second inhibitor. Therefore, it should be stressed that antiretroviral treatments including PR inhibitors should aim at a full suppression of viral replication in vivo, thereby preventing any cryptic selection of resistant and potentially cross-resistant variants.
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ACKNOWLEDGMENTS |
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We thank Fabrizio Mammano and Esther Race for their interest in this work and Luc Montagnier, Pierre Galanaud, and Jean Dormont for their support.
This work was supported in part by grants from the Agence Nationale de Recherches sur le Sida (ANRS).
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Recherche Antivirale, IMEA-INSERM, Hôpital Bichat-Claude Bernard, 46, rue Henri Huchard, 75018 Paris, France. Phone: 33 1 40 25 63 63. Fax: 33 1 40 25 87 80. E-mail: clavel{at}bichat.inserm.fr.
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