ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2008, p. 1545–1548
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 52, No. 4
In Vitro Phenotypic Susceptibility of Human Immunodeficiency Virus
Type 2 Clinical Isolates to Protease Inhibitors?
Delphine Desbois,1Be ´ne ´dicte Roquebert,1Gilles Peytavin,2Florence Damond,1Gilles Collin,1
Antoine Be ´nard,5Pauline Campa,4Sophie Matheron,3Genevie `ve Che ˆne,5
Franc ¸oise Brun-Ve ´zinet,1and Diane Descamps1* for the
French ANRS HIV-2 Cohort (ANRS CO 05 VIH-2)
Laboratoire de Virologie, Service de Microbiologie, Ho ˆpital Bichat-Claude Bernard, Paris, France1; Laboratoire de Toxicologie,
Service de Pharmacie, Ho ˆpital Bichat-Claude Bernard, Paris, France2; Service de Maladies Infectieuses et Tropicales,
Ho ˆpital Bichat-Claude Bernard, Paris, France3; Service de Maladies Infectieuses et Tropicales,
Ho ˆpital Saint-Antoine, Paris, France4; and ISPED, Universite ´ Victor Segalen Bordeaux 2,
INSERM U593, Bordeaux, France5
Received 4 October 2007/Returned for modification 19 November 2007/Accepted 14 January 2008
We determine phenotypic susceptibility of human immunodeficiency virus type 2 (HIV-2) isolates to am-
prenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, saquinavir, and tipranavir. Saquinavir, lopi-
navir, and darunavir are potent against wild-type HIV-2 isolates and should be preferred as first-line options
for HIV-2-infected patients. Other protease inhibitors are less active against HIV-2 than against HIV-1.
Few data are available on human immunodeficiency virus
type 2 (HIV-2) susceptibility to antiretroviral agents. In
France, as recommended by the national expert group on the
treatment of HIV infection, HIV-2-infected patients receive
highly active antiretroviral therapy (HAART) regimens as
HIV-1-infected individuals do, except without nonnucleoside
reverse transcriptase inhibitors or fusion inhibitor classes (17,
12). However, a recent study showed that CD4 cell recovery
was poor in antiretroviral-naive HIV-2-infected patients start-
ing treatment with HAART (8). Thus, it appears crucial to
determine HIV-2 susceptibility to the current protease inhib-
itors (PIs) available in order to define the optimal regimen to
(This work was presented at the 14th Conference on Retro-
viruses and Opportunistic Infection, Los Angeles, CA, 25 to 28
We selected nine PI-naive HIV-2-infected patients from the
French HIV-2 ANRS cohort. Six of these patients subse-
quently received HAART regimens including a PI (indinavir,
nelfinavir, saquinavir, and/or ritonavir) for a median of 13
months (range, 2 to 36) and had plasma viruses harboring
mutations in the protease gene. Peripheral blood mononuclear
cell (PBMC) coculture isolates were collected from these pa-
tients before (T0, n ? 6) and during (T1 [time of collection of
first plasma specimen during PI exposure], n ? 6; T2 [time of
collection of second plasma specimen during PI exposure], n ?
2) PI exposure. Protease gene sequences of plasma and PBMC
isolates were determined as previously described (3). Amino
acid changes were compared with those associated with drug
resistance in HIV-1 (International AIDS Society-USA [IAS-
USA]). We determined the in vitro phenotypic susceptibility of
clinical HIV-2 isolates and HIV-2 reference strain ROD and
HIV-1 reference strain BRU to amprenavir, atazanavir, da-
runavir, indinavir, lopinavir, nelfinavir, saquinavir, and tiprana-
vir using the ANRS PBMC assay (4). Phenotypic inhibitory
quotients (PIQs) were calculated for each clinical HIV-2 iso-
late as the ratio between the trough plasma PI concentration
and the 50% inhibitory concentration (IC50). The PIQs were
not adjusted for protein binding. Table 1 shows the phenotypic
results and protease gene sequences of the HIV-2 clinical
isolates and the phenotypic results of the HIV-2 and HIV-1
The protease sequences of the wild-type HIV-2 strains, com-
pared to the HIV-1 clade B consensus sequence, contained
several amino acids associated with HIV-1 PI resistance, such
as 10I/V, 16E, 20V, 32I, 33V, 35G, 36I, 43T, 46I, 47V, 58E,
62V, 69K, 71V, 73A, 82I, and 93L. Other differences were
observed at positions involved in but not associated with HIV-1
resistance, such as 13A, 34A, 60K, 63E, 76M, 77T, 85F, and
89I. Relative to the HIV-1 reference strain, the median IC50
values of the HIV-2 wild-type isolates were 31-fold higher for
amprenavir, eightfold higher for atazanavir, sevenfold higher
for tipranavir, and threefold higher for indinavir and nelfinavir.
Darunavir, lopinavir, and saquinavir median IC50and IC90
values were similar for HIV-1 and the wild-type HIV-2 isolates
(Table 1). Viruses isolated from the six PI-experienced pa-
tients at T1 and T2 harbored the I82F, I84V, and L90M sub-
stitutions, alone or in combination with minor HIV-1 PI mu-
tations, such as V10I, V33I, I54M, I64V, V71I, and I89V.
Compared to the corresponding wild-type isolates, the six mu-
tants showed increases of fourfold to ?10-fold in the IC50
and/or IC90values of all tested PIs, at both T1 and T2. The PIQ
values of amprenavir, atazanavir, indinavir, nelfinavir, and
tipranavir were, respectively, 33-fold, eightfold, threefold,
threefold, and sevenfold lower for HIV-2 wild-type strains than
for HIV-1. Darunavir, lopinavir, and saquinavir PIQ values
* Corresponding author. Mailing address: Laboratoire de Virologie,
Ho ˆpital Bichat Claude Bernard, 46 rue Henri Huchard, 75018 Paris,
France. Phone: 33 1 40 25 61 54. Fax: 33 1 40 25 67 69. E-mail:
?Published ahead of print on 28 January 2008.
PIs are designed to fit the active site of the HIV-1 protease
and are sensitive to structural changes in the viral protein. It
has been reported that the therapeutic outcome of HIV-2-
infected patients might be influenced by the choice of PIs (1,
15). For amprenavir, our data are in keeping with those re-
ported elsewhere, showing significantly less activity against
HIV-2 wild-type strains than against HIV-1 (9, 14, 16). These
phenotypic results could be explained by the natural presence
in HIV-2 protease of amino acids associated with resistance to
HIV-1, which might influence the binding affinity of the PIs for
HIV-2 protease (2, 3, 9, 10, 11). In our study, all the HIV-2
wild-type strain protease sequences naturally presented the
amino acids 32I and 47V, associated with resistance in HIV-1
infection to amprenavir, according to the IAS-USA list and to
different genotypic resistance interpretation algorithms (www
.hivfrenchresistance.org, http://hivdb.stanford.edu and www
.kuleuven.be/rega/cev/links/rega_algorithm). We found that
clinical HIV-2 isolates and HIV-1 reference strains had similar
phenotypic susceptibility to saquinavir, lopinavir, and darunavir.
As amprenavir and darunavir are structurally close, we expected
darunavir to be relatively ineffective in HIV-2. Crystallographic
studies with HIV-1 showed that darunavir interacts directly
with the main chains of aspartic acid residues (Asp-29 and
Asp-30), whereas other PIs interact with side chains in the S2
subsite of the HIV-1 enzyme (6, 7). Moreover, it has been
reported for HIV-1 that the binding affinity of darunavir for
the wild-type protease was ?100-fold higher than those of
other PIs due to the slower dissociation rate of this molecule
TABLE 1. Phenotypic susceptibilities to PIs and/or protease mutations of the HIV-2 subtype A and B consensus sequences of the nine
wild-type isolates and HIV-1 and HIV-2 reference isolates before (T0) and during (T1 and T2) PI treatmenta
HIV-1 reference isolate BRU
HIV-2 reference isolate ROD
HIV-2 clinical isolates from:
Patient 1 (subtype H)
Patient 2 (subtype A)
Patient 3 (subtype A)
Patient 4 (subtype A)
Patient 5 (subtype A)
Patient 6 (subtype B)
Patient 7 (subtype B)
Patient 8 (subtype B)
Patient 9 (subtype A)
0.90 2.70 0.10 0.500.0040.040.020.50
10.0 (12)82.0 (30)1.90 (16)15.0 (32) 0.90 (225)4.70 (126) 12.0 (621) 99.0 (193)
9.50 (22)31.0 (9)0.20 (2)1.80 (6) 0.80 (216)3.00 (88)0.20 (15)3.10 (28)
12.0 (36)96.0 (60) 0.04 (0.8)1.30 (6) 0.50 (124)2.40 (105) 6.40 (400) 51.0 (143)
12.0 (35) 95.0 (60) 0.40 (9)3.00 (14)1.00 (254) 8.10 (351) 11.0 (705)90.0 (252)
30.0 (50) 110 (24)
17.0 (30)140 (30)0.20 (3)1.40 (4)0.70 (324)5.10 (429)12.0 (100)95.0 (161)
0.402.400.20 0.40 0.0040.02 0.0040.02
0.803.30 0.030.300.100.500.006 0.20
Median IC50/IC90value of
clinical isolates at
0.60 (?0.20)3.30 (?1.50)0.06 (?0.05)0.30 (?0.08)0.004 (?0.04)0.03 (?0.16)0.02 (?0.10)0.40 (?0.80)
aValues in parentheses are the fold increases over the values for T0.
bProtease gene sequences are given for the indicated time points. *, substitutions selected between T0 and T1/T2.
1546NOTESANTIMICROB. AGENTS CHEMOTHER.
from the protease active site (5). In the same way, crystallog-
raphy structure studies of the HIV-2 protease and binding
affinity experiments might help us to understand the difference
observed in the natural susceptibilities of HIV-2 strains to
these two drugs as well as phenotypic resistance in HIV-2
mutated strains. The IC50and IC90values of atazanavir, indi-
navir, nelfinavir, and tipranavir for the HIV-2 isolates were
higher than those observed for HIV-1, suggesting the hypoth-
esis of the lower activities of these PIs against HIV-2. How-
ever, these values were lower than their respective trough
plasma concentrations. This might be explained by the fact that
HIV-2 wild-type isolates harbored several amino acids associ-
ated with PI resistance in HIV-1.
PI treatment-associated amino acid changes in the HIV-2
protease gene occurred at positions known to confer PI resis-
tance in HIV-1 and were not associated with the use of a
particular PI without any order of accumulation. They altered
the phenotypic susceptibility of the isolates to all the PIs tested
here. These results are in keeping with data published else-
where (2, 3, 9, 11, 13, 15). Mutagenesis experiments coupled
with phenotypic susceptibility testing might help to determine
the impact of each substitution on PI resistance. Saquinavir,
lopinavir, and darunavir appear to be the best choices for
first-line therapy of HIV-2 infection, while amprenavir should
not be used. Atazanavir and tipranavir might be used with care
(17). Our results suggest that treatment guidelines for HIV-1-
infected patients should not be directly extrapolated to HIV-
2-infected patients. Virological efficacy data in vivo might help
us to evaluate the place of PIs in HIV-2 antiretroviral strategy.
Drugs and sources. Amprenavir was provided by Glaxo-
SmithKline (Marly-le-Roi, France), atazanavir by Bristol-
Myers Squibb (Rueil-Malmaison, France), darunavir by Tibotec
Lopinavir Nelfinavir SaquinavirTipranavir
3.20 (57)8.70 (17) 9.00 (236)24.0 (79) 7.30 (252) 31.0 (114)7.30 (19)16.0 (20)
26.0 (12)110 (450)
1.70 (75)8.50 (42)1.20 (24)15.0 (37)0.09 (45)0.70 (33)4.10 (12)23.0 (9)
0.10 (5)0.80 (10)
?17.6 (/)6.40 (1,287)51.0 (2,221)2.80 (8)16.0 (4)
0.10 (7)1.10 (14)10.0 (66) 81.0 (193)22.0 (4,421) 180 (7,635)4.80 (13)29.0 (7)
17.0 (10)107 (107)
0.10 (3)0.9 (12)
?17.6 (/)6.70 (0.5) 52.0 (0.6)4.00 (10) 12.0 (7)
0.03 0.100.060.200.007 0.020.40 3.00
0.020.20 0.040.80 0.060.300.30 2.60
0.040.100.202.10 0.020.200.30 0.7
0 0.03 (?0.01)0.14 (?0.10)0.06 (?0.08)0.40 (?0.60) 0.008 (?4.60)0.05 (?27.0) 0.30 (?0.05)2.50 (?1.10)
VOL. 52, 2008NOTES 1547
(Mechelen, Belgium), indinavir by Merck Sharp & Dohme-
Chibret (West Point, PA), lopinavir by Abbott (Rungis,
France), nelfinavir and saquinavir by Roche (Neuilly sur Seine,
France), and tipranavir by Boehringer-Ingelheim (Ridgefield,
This work was supported by Agence Nationale de Recherche sur le
SIDA et les He ´patites Virales (ANRS).
We thank Laetitia Stephant for her technical skills.
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1548NOTESANTIMICROB. AGENTS CHEMOTHER.