Most areas with high levels of HIV seroprevalence also have endemic malaria. Chloroquine (CQ) remains the antimalarial drug most widely used in the world, despite the increasing diffusion of drug-resistant Plasmodium falciparum strains. More information is therefore needed on the effects of CQ on HIV replication and on the interactions of CQ with antiretroviral drugs.
CQ and its hydroxy-analogue hydroxychloroquine (HCQ) are endowed with anti-HIV effects, inhibiting the production of infectious viral particles at doses nontoxic for human cultured cells. 1–4 2 3 2 3 5
CQ/HCQ has an in vitro and in vivo anti-HIV effect similar to zidovudine (AZT) alone and exerts an additive effect with the combination of either didanosine or AZT and hydroxyurea. 6–9 1,10 11–17
The aims of the present study are (1) to clarify further the mechanisms of HIV inhibition by CQ and (2) to determine the in vitro effects of CQ in combination with PIs on HIV replication and on P-gp and MRP1 function.
MATERIALS AND METHODS
Drugs
Chloroquine and AZT were purchased from Sigma (St. Louis, MO). Among the HIV PIs, we chose the first-generation PIs indinavir (IDV), ritonavir (RTV), and saquinavir (SQV), because their cellular effects are the best studied and they will probably be the first PIs available on a large scale in resource-poor areas. The PIs were kindly provided by the manufacturers (IDV: Merck Sharp and Dohme Ltd, Hoddesdon, Hertfordshire, UK; SQV: Roche Registration Ltd, Welwyn Garden City, Hertfordshire, UK; and RTV: Abbott Laboratories Ltd, Queenborough, Kent, UK). They were dissolved in dimethyl sulfoxide (DMSO; maximum content = 0.5% vol/vol).
Description of the Viruses Employed and Infection Assays
We used both laboratory-adapted strains and primary isolates. The laboratory-adapted HIV-1IIIB and HIV-2CBL/20 strains and the primary isolates HIV-1UG3 (clade A, R5) and HIV-1VI 829 (clade C, R5) from antiretroviral-naive African subjects were obtained as previously described. 2,18 PAVIA11 and HIV-1PAVIA12 (clade B, coreceptor usage undetermined) were isolated from Italian individuals with highly active antiretroviral therapy (HAART) failure and possess a genotypic profile of multidrug resistance. 19 PAVIA11 and L10I, M46L, I54V, L63V, A71T, V82A, and I93L in HIV-1PAVIA12 , whereas RT resistance mutations were M41L, E44D, D67N, T69D, K103N, V108I, V118I, M184V, L210W, T215Y, F227FL, and K238KT in HIV-1PAVIA11 and M41L, E44D, D67N, M184V, L210W, and T215Y in HIV-1PAVIA12 . HIV-1SQV-resistant (clade B, X4) has in vitro SQV-induced PI resistance 20 14 A018-G910-6 and HIV-18 A012-G691-6 were isolated in the late 1980s from human subjects after AZT monotherapy. 21 14 A018-G910-6 displayed M41L, D67N, K70R, T215Y, and K219Q, and HIV-18 A012-G691-6 displayed D67N, K70R, V118I, T215F, and K219Q. Viral stocks were obtained and titrated as described elsewhere. 2,18
HIV-genotype analysis was performed on the same viral stock suspensions as used for the acute infection experiments by means of a commercially available kit (Abbott). In brief, viral RNA was extracted by the QIAmp Viral RNA kit (Qiagen, Valencia, CA), retrotranscribed by murine leukemia virus RT, and amplified with amplitaq-Gold polymerase enzyme (Applied Biosystems, Foster City, CA) by using 2 different sequence-specific primers for 40 cycles. 22 Pol -amplified products (containing the entire protease and the first 324 amino acids of the RT open reading frame) were full-length sequenced in sense and antisense orientations by using 7 different overlapping sequence-specific primers by an automated sequencer (ABI 3100; Applied Biosystems). 22
In acute infection assays, the different cell types were incubated at 37°C for 2 hours with the viral stock suspensions at a multiplicity of infection (MOI) of approximately 0.1, unless otherwise specified. After 3 washes, cells were incubated in fresh culture medium for 7 days at 37°C, and cell-free supernatants at different intervals after infection were harvested for enzyme-linked immunosorbent assay (ELISA) measurement of HIV-1 p24 (NEN Life Science Products, Boston, MA) or HIV-2 p27 (Coulter, Hialeah, FL). 2 6 2
The CD4+ CXCR4+ MT-4 T-cell line was used to assess the effects of CQ and PIs on the X4 laboratory-adapted strains, whereas peripheral blood mononuclear cells (PBMCs) obtained by informed consent from healthy donors and stimulated for 3 days with 2 μg/mL of phytohemagglutinin (PHA; Difco Laboratories, Detroit, MI) were adopted in assays using primary isolates.
To measure synergism, cell pellets were resuspended in media containing different combinations of CQ and IDV after viral adsorption onto cells. A fractional inhibitory concentration (FIC) was then calculated as the ratio: 90% effective concentration (EC90 ) of drug A in combination with drug B / EC90 of drug A alone. The effect was considered to be synergistic when the sum of FICs was ≤0.5.
Assays for Evaluation of Toxicity
Cell proliferation, viability, and apoptosis were analyzed by [3 H] thymidine incorporation, by the methyl-tetrazolium (MTT) method, and by propidium iodide/annexin V fluorescein isothiocyanate (FITC) staining, respectively, as determined by techniques previously validated in our laboratories. 23–25
Measurement of HIV Glycosylation
Lymphocytic H9 cells were infected with HIV-1IIIB or HIV-2CBL/20 and then plated (6 × 105 cells per well) in macrowell cultures (35-mm diameter; Falcon, Oxnard, CA). We used H9 cells rather than MT-4 cells, the latter being constitutively infected with HTLV-1. After 3 days, cells were treated with different concentrations of CQ (0.1–1000 μM) for 1 hour to achieve steady-state conditions and subsequently labeled with [3 H] glucosamine. For labeling, the medium was discarded and the cells were washed 3 times and incubated with RPMI-1640 containing 5% fetal calf serum (FCS), penicillin/streptomycin, and [3 H] glucosamine (1 μCi/mL; Amersham, Arlington Heights, IL). The labeling medium was removed after 16 hours. Virus was isolated from the supernatant of the H9 treated and untreated cells by ultracentrifugation in sucrose density gradients using a K-6 rotor with a 20% to 60% (wt/vol) sucrose gradient. The supernatant containing the virus was precipitated with 10% trichloroacetic acid (TCA; Sigma) and passed over a glass fiber filter (particle retention of 1.2 μm; Whatman, Maidstone, UK). The filter was washed 3 times with 5% vol/vol TCA, dried with 100% methanol, and counted in a scintillation counter. 26
Internal Labeling With [35 S] Cysteine and [35 S] Methionine and Immunoprecipitation
The same HIV-1IIIB –infected H9 cells used for [3 H] glucosamine labeling were washed 3 times after loading with CQ, as described previously. The cells were then resuspended at a density of 107 cells/mL in cysteine- and methionine-free RPMI-1640 (Sigma) plus 10% dialyzed FCS, 1% glutamine, and antibiotics. The cells were then metabolically labeled with [35 S] cysteine and [35 S] methionine (NEN) at 50 μCi at a rate of 5 × 106 cells for 16 hours. After incubation, the viable cells were counted again. The cell suspensions were then centrifuged, and the supernatants were subjected to immunoprecipitation with human anti-gp120 antibodies as previously described. 25
Determination of P-Glycoprotein and Multidrug Resistance Protein-1 Expression
For P-gp immunodetection, peripheral blood lymphocytes (PBLs) were incubated with the mouse monoclonal antibody (mAb) MRK-16 (Kamiya Biomedicals Company, Seattle, WA) as previously reported. 5
Flow Cytometric Detection of P-Glycoprotein and Multidrug Resistance Protein-1 Efflux Activity
For study of P-gp function, we used the dihydrorhodamine 123 (Rh123) efflux assay as previously reported. 27 28–30 2 . The cells were then washed in ice-cold phosphate-buffered saline (PBS), and Rh123 or CF accumulation was determined using a FACScan flow cytometer (Becton Dickinson). For efflux experiments, cells were resuspended in Rh123- or CF-free medium with or without drugs and incubated for 60 minutes at 37°C, 5% CO2 , and then washed in ice-cold PBS. For dual and triple fluorescence, Rh123- or CF-stained cells were counterstained with phycoerythrin (PE)- and peridinin chlorophyll protein (PerCP)–conjugated mAbs. The mean retained intracellular CF fluorescence intensity was then estimated using flow cytometry as for the accumulation experiments. In general, FACS data were expressed as mean fluorescence intensity (MFI), where the fluorescence intensity distribution was approximately Gaussian on a linear scale, or as the percentages of Rh123bright /dim and CFbright /dim cells, where the fluorescence intensity distribution was bimodal.
RESULTS
Chloroquine Inhibits HIV-1 and HIV-2 Replication by Impairing Virus Glycosylation
The effects of CQ/HCQ on HIV replication have been studied by 2 different approaches: (a) by loading cells for 1 hour with CQ/HCQ concentrations mimicking those observed in lymphocytes of individuals under chronic treatment with CQ/HCQ 1,6 2
In a first step, we confirmed that CQ dose dependently inhibited HIV-1 and HIV-2 replication either in H9 cells treated with method a (HIV-1:P < 0.01, HIV-2:P < 0.05, t test for regression) or in MT-4 cells treated with method b (HIV-1:P < 0.05, HIV-2:P < 0.05, t test for regression;Fig. 1A ). Moreover, both treatments at any of the CQ concentrations tested caused no increases in cell death or apoptosis or inhibition of cellular proliferation as determined by MTT and propidium iodide staining as well as by [3 H] thymidine incorporation (not shown). We conclude that CQ efficiently and dose dependently inhibits HIV-1 and HIV-2 replication in the absence of toxic effects independent of the CQ administration method. These data reconcile the different methods used in the past to evaluate the anti-HIV effects of CQ, even if the intracellular concentrations of CQ reached by both methods are likely to differ because of differences in the cell lines of choice. These results allowed us to use either method in the present study.
FIGURE 1.:
Anti-HIV effects of chloroquine (CQ). A, Concentrations of CQ inhibiting the 50% of viral replication (EC50 ) and EC90 in acutely infected H9 cells preloaded with different CQ concentrations (method a) and in MT-4 cells constantly incubated with different CQ concentrations after viral adsorption onto cells (method b). B, Effects of CQ on HIV-1 glycosylation. C, Effects of CQ on the peptidic portion of the HIV-1 gp120 envelope glycoprotein. In (A), viral replication was measured 4 days after infection with HIV-1IIIB or HIV-2CBL/20 at a multiplicity of infection of 0.1. Results are presented as the EC50 and EC90 of CQ. Values represent medians from 3 independent experiments (ranges were <20% of the values reported). The method followed (method a or method b) is indicated in parentheses. In (B), 106 HIV-1IIIB –infected cells were loaded with CQ according to method a and incubated for 16 hours with [3 H] glucosamine to label the carbohydrates. Virus was isolated from the supernatants and measured for 3 H activity in a scintillation counter. Results (mean ± SD from 3 experiments) are expressed as counts per minute and are normalized by the reverse transcriptase activity of supernatants from which the virus was isolated. In (C), 107 HIV-1IIIB –infected cells were loaded with CQ according to method a and incubated for 16 hours with [35 S] cysteine/methionine to label the peptidic portion of gp120. The supernatants were then subjected to immunoprecipitation with anti-gp120 antibodies. The immunoprecipitated material was then separated by electrophoresis on a 10% polyacrylamide gel and visualized by autoradiography. One representative experiment of 3 experiments.
Previous studies indicated that CQ impairs the formation of the heavily glycosylated epitope 2G12 on gp120 and excluded other mechanisms of HIV inhibition by CQ/HCQ. 2,6 3 H] glucosamine. The 3 H signal in precipitated HIV-1IIIB particles was decreased in a dose-dependent manner by CQ (P < 0.05, t test for regression; see Fig. 1B ). Similar results were obtained with HIV-2CBL/20 (not shown). In contrast, CQ (up to 1 mM) did not inhibit incorporation of [35 S] cysteine/methionine into gp120 (see Fig. 1C ), thus confirming results previously obtained. 2
Chloroquine Increases HIV Inhibition in the Presence of Indinavir
Because CQ inhibits a posttranslational step in the HIV life cycle, we tested its effects in combination with a PI. We first investigated whether the addition of CQ to IDV might result in increased inhibition of HIV-1 replication compared with IDV alone. To test this hypothesis, HIV-1IIIB –infected MT-4 cells were incubated with 10 nM of IDV in the presence or absence of increasing concentrations of CQ (1–6.25 μM according to method b). The IDV concentration chosen is close to the 50% effective concentration (EC50 ) in HIV-1IIIB –infected MT-4 cells as determined under our experimental conditions (not shown). Addition of CQ (0.78–6.25 μM) to IDV caused no significant impairment of cell viability (not shown) but significantly decreased the levels of HIV-1 p24 at day 5 after infection (P < 0.05, repeated-measures ANOVA;Fig. 2 ). This decrement was not dose dependent (P = 0.7, t test for regression) as shown by the loss of the CQ effects in the cell cultures treated with a clinically unreachable concentration of the antimalarial (12.5 μM). Such absence of dose dependence with the IDV/CQ combination differs from the dose-dependent anti-HIV effect (P < 0.05, t test for regression) observed with CQ alone (see Fig. 2 ).
FIGURE 2.:
Combined effects of chloroquine (CQ) and indinavir (IDV) on HIV-1 replication. MT-4 cells were inoculated with HIV-1IIIB and subsequently incubated with 10 nM of IDV with or without increasing CQ concentrations. In this case, cells were infected with a low multiplicity of infection (0.01) to better unmask any favoring effects of the IDV/CQ combination on HIV-1 replication. Viral replication is shown as the level of HIV-1 p24 in the culture medium at day 5 after infection (mean ± SEM in 3 experiments). The effects of CQ in the absence of IDV are also shown.
Chloroquine Exerts a Synergistic Anti-HIV Effect in Combination With Protease Inhibitors
To explore this phenomenon better, we investigated whether the effects of different CQ/IDV combinations were additive, synergistic, or subsynergistic. HIV-1IIIB –infected cells were treated with various concentrations of CQ (according to method b) or IDV alone or in combination. We then determined which concentrations of each drug in the different combinations produced 90% inhibition of HIV-1 replication. For each drug, we calculated the FIC. Analysis using the isobologram method showed that the effect of CQ on the anti–HIV-1 activity of IDV was synergistic at the low FICs of CQ (corresponding to prophylactic antimalarial plasma concentrations, ie, ≃0.1–1 μM), 31 Fig. 3A ). Similar effects were obtained with HIV-2CBL/20 (not shown) and using CQ in combination with RTV or SQV (Fig. 3B ). We conclude that concentrations of CQ as found during malaria prophylaxis exert a synergistic anti-HIV effect in combination with PIs.
FIGURE 3.:
Combined effects of chloroquine (CQ) and HIV protease inhibitors (PIs) on viral replication. A, Isoboles showing the fractional inhibitory concentrations (FICs) of the combination of CQ plus indinavir (IDV) against HIV-1IIIB replication. B, Isoboles showing the FICs of the combination of CQ with saquinavir (SQV) or with ritonavir (RTV) against HIV-1IIIB replication. (C,D) Partial restoration by CQ (1 μM) of the response to PIs of the HIV-1PAVIA11 (C) and HIV-1PAVIA12 (D) multidrug resistant isolates in peripheral blood mononuclear cells (PBMCs). Briefly, MT-4 cells or primary PBMCs were inoculated with laboratory strains or primary isolates, respectively, at a multiplicity of infection of 0.1. The HIV-infected cells were then incubated with selected concentrations of PIs and/or CQ. Panels (A) and (B) show the FICs of the CQ/PI combination capable of inhibiting viral replication by 90% as calculated on the basis of 3 independent experiments (ranges were <20% of the values reported). The curves best fitting the data points were calculated according to a nonlinear regression model. The graphs show the expected line in case of simply additive effects (the straight line connecting the 1.0 FIC values on the x and y axes) as well as the threshold between subsynergism and true synergism (ie, the dotted line connecting the 0.5 FIC values). In panels (C) and (D), the data points (mean ± SEM) show the replication rates of the viral isolates in the presence of IDV or SQV in the presence or absence of CQ (1 μM). Results are expressed as a percentage of the viral replication levels obtained in the absence of drugs. The regression lines best fitting the data points demonstrate the CQ-induced restoration of a dose-dependent HIV inhibition by PIs. The results of 3 experiments using PBMCs from 3 different donors are shown (mean ± SEM in 3 experiments).
Chloroquine Increases the Susceptibility of Primary HIV Isolates to Protease Inhibitors
We then tested the effects of CQ on the susceptibility to PIs of PI-sensitive and resistant primary HIV-1 isolates and of a PI-resistant laboratory strain, (HIV-1SQV-resistant ;Table 1 ). For this purpose, MT-4 cells or PHA-stimulated PBMCs were infected with the viruses, washed, and incubated with increasing concentrations of IDV or SQV in the presence or absence of 1 μM of CQ.
TABLE 1:
We found that CQ not only decreased the PI's EC50 values in PI-susceptible isolates but in 3 PI-resistant viruses as well (P < 0.05, Wilcoxon rank sum; see Table 1 ), partially restoring PI sensitivity in viruses such as HIV-1PAVIA11 and HIV-1PAVIA12 (HIV-1PAVIA11 :P < 0.01, HIV-1PAVIA12 :P < 0.05, t test for regression), which display no dose-dependent response to PIs in the absence of CQ (HIV-1PAVIA11 :P = 0.660, HIV-1PAVIA12 :P = 0.583, t test for regression; see Figs. 3C, D ). The effects of the CQ/PI combination were synergistic, the sum of FICs being ≤0.5 in the 3 sets of experiments allowing this calculation (see Table 1 ). This result cannot be attributed to toxic effects of the CQ/IDV combination, because the PBMC viability was similar whether cell treatment consisted of IDV with or without 1 μM of CQ (not shown).
In contrast, CQ had no effect on the response to AZT in AZT-resistant isolates (see Table 1 ). In these experiments, the sum of FICs of the CQ/AZT combination lay within values of 1 to 1.2, indicating an additive but not a synergistic effect in keeping with previous studies. 6,9
Chloroquine Enhances the Inhibitory Effect Exerted by Protease Inhibitors on the P-Glycoprotein– and Multidrug Resistance Protein-1–Mediated Efflux Activity
Because both CQ and PIs inhibit P-gp and MRP1, 14–17,24 27–30
To confirm the effects of CQ on P-gp– and MRP1-mediated efflux, intracellular retention of Rh123 and CF was determined in normal human PBLs in the presence or absence of various concentrations of CQ. CQ was found to enhance Rh123 and CF accumulation by inhibiting their efflux in a dose-dependent manner (Fig. 4 ). PIs alone at concentrations of 3 μM had little or no inhibitory effect on Rh123 efflux, depending on the baseline efflux activity of the donor and on the PI adopted (ritonavir > saquinavir ≃ indinavir). These effects are milder than those observed using higher PI concentrations (10 μM), which produce more sustained inhibition of Rh123 efflux, which is evident in PBLs from all donors and with all the PIs adopted. 27 P < 0.05, Kolmogorov-Smirnov statistics; see Fig. 4 ). These effects were highly reproducible in lymphocytes from all donors tested (n = 4). Three-color immunofluorescence demonstrated that the IDV/CQ combination significantly (P < 0.05, Kolmogorov-Smirnov statistics) inhibited Rh123 and CF efflux not only in the memory but in the naive CD4+ subpopulation showing higher efflux activity (Fig. 5 ). We conclude that CQ increases the blockade of P-gp– and MRP1-mediated efflux activity exerted by PIs.
FIGURE 4.:
Effect of various concentrations of chloroquine (CQ; 1 μM and 3 μM) and of protease inhibitors (3 μM) on intracellular accumulation and efflux of rhodamine (Rh123; right panels) and carboxyfluorescein (CF; left panels) in normal peripheral blood lymphocytes (PBLs). Cells were stained with Rh123 (specific substrate for P-glycoprotein [P-gp] or CF (specific substrate for multidrug resistance protein-1 [MRP1]). To estimate Rh123 and CF uptake, P-gp and MRP-1 activity was blocked by transferring cells to 4°C immediately after staining with Rh123 and CF. P-gp and MRP1 function was inferred from a decrease in fluorescence after incubation in Rh123- and CF-free medium for 1 hour. CQ dose dependently inhibited Rh123 and CF efflux. Indinavir (IDV; 3 μM), saquinavir (SQV; 3 μM), or ritonavir (RTV; 3 μM) further increased the retention of the dyes. The fluorescence histograms illustrate 1 of 4 experiments with similar results using PBLs from 4 different donors. The number of events is indicated by the y axis. Note that because of the different fluorescence distribution, unimodal for CF and bimodal for Rh123, results are expressed as mean CF fluorescence intensities and percentages of Rh123dim/bright cells, respectively. The latter were established by means of a cursor (vertical line) positioned on histograms referring to control uptake samples.
FIGURE 5.:
Effect of chloroquine (CQ) on rhodamine (Rh123) and carboxyfluorescein (CF) efflux from CD45RA
+ naive (A) and CD45RO
+ memory (B) CD4
+ cells in the presence or absence of indinavir (IDV). Peripheral blood mononuclear cells from a healthy control subject were stained with peridinin chlorophyll protein–conjugated monoclonal antibodies (mAbs) directed against CD4 and with phycoerythrin-conjugated mAbs to CD45RA and CD45RO. P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP1) function was inferred, respectively, from a decrease in fluorescence of Rh123- and CF-loaded cells after 1 hour of incubation in fluorochrome-free media. Inhibitory effects on Rh123 and CF efflux were observed in the presence of CQ (1 μM), and higher inhibition rates were observed in the presence of CQ plus IDV (3 μM). Rh123 and CF uptake was measured as explained in the legend of
Figure 4 . Events were gated on side scatter and CD4
+ fluorescence. The population of interest was further defined on the basis of the CD45 isoform. The histograms refer to the fluorescence intensity of the cell subpopulations of interest. The number of events is indicated by the
y axis. Histograms represent 1 of 3 experiments with similar results using lymphocytes from 3 different donors.
Finally, we examined the impact of CQ and IDV on P-gp and MRP1 expression. Our results confirm the previously reported lack of effect of PIs on P-gp and MRP1 expression. 27
DISCUSSION
The results of this study show (1) that CQ inhibits viral particle glycosylation and (2) that CQ in combination with PIs carries out synergistic anti-HIV effects and combined inhibitory effects on P-gp and MRP1 function.
CQ inhibits HIV-1 and HIV-2 replication by a dose-dependent mechanism as shown (a) by acute (1 hour) CQ loading of cells using high concentrations of the drug to reach steady-state conditions 6 2,3 50 /EC90 values are apparently 10- to 100-fold lower in MT-4 cells treated with method b than in H9 cells loaded with method a; this discrepancy can be reconciled in light of the kinetics of intracellular accumulation of CQ. 32
We previously demonstrated that the sole mechanism explaining the anti-HIV activity of CQ is a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2G12 epitope of gp120. 2 33 1,34 2
The effect of CQ on HIV glycosylation may account for its effects in combination with a major class of antiretroviral drugs (ie, the PIs). In this article, we show that the addition of increasing CQ concentrations to IDV results in increased inhibition of HIV-1 replication compared with IDV alone. This decrement is not dose dependent as opposed to that observed using CQ alone. The lack of dose dependency is explained by the isobologram analysis of the results obtained with various concentrations of CQ or PIs alone or in combination. According to this analysis, the combined effect of CQ and a PI is synergistic at the lowest CQ concentrations, additive at higher CQ concentrations, and antagonistic at suprapharmacologic concentrations of the antimalarial drug. The loss of a synergistic effect paralleling the increase in CQ concentrations thus accounts for the lack of a dose-dependent HIV inhibition by CQ in the presence of IDV. Interestingly, the synergistic activity of CQ/PIs was observed using a CQ concentration of 0.1 to 1 μM, which is similar to the CQ concentrations found in the plasma of individuals receiving malaria prophylaxis. 31 3,10 35 9
CQ increases P-gp and MRP1 blockade by PIs, which was recently postulated to have important consequences for the intracellular concentrations of antiretrovirals. 11–14,27 + lymphocytes and was also evident in naive subpopulations, which have higher P-gp– and MRP1-mediated efflux activity than memory cells. Interestingly, the combined CQ/PI effects were more evident on MRP1, whose efflux activity was recently suggested by Speck et al 36 36
A major concern regarding the effects of CQ on P-gp and MRP1 is the possibility of an upregulation of these proteins. In the present study, the increased anti–P-gp– and anti-MRP1–antibody binding capacity of cells treated with CQ for 48 hours was not associated with increased Rh123 and CF efflux, suggesting a conformational change of both P-gp and MRP1 because of the presence of CQ rather than a true induction of their expression. This hypothesis is in line with observations of others reporting that (1) substrates induced conformational changes in surface pumps, increasing binding of specific antibodies, 37,38 39 40
In conclusion, this study provides the first evidence of synergistic anti-HIV activity between CQ and PIs. These in vitro results warrant in vivo confirmation in HIV-positive individuals by comparing the long-term antiviral effect of PI-containing HAART regimens with and without CQ or HCQ. If such studies provide confirmatory results, they may lead to a strategy whereby coadministration of CQ/HCQ allows lowering of the PI dosage, lessening cost and possibly toxicity. Cost lessening would of course be particularly welcome in developing countries. Moreover, the potential ability of CQ to overcome resistance to PIs could be important in the treatment of drug-experienced HIV-positive subjects who have developed multiple resistances to antiretroviral drugs and thus have limited therapeutic options.
ACKNOWLEDGMENTS
The authors thank the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD), for providing HIV-2CBL-20/H9 , HIV-1SQV-resistant , HIV-114 A018-G910-6 and HIV-18 A012-G691-6 ; Maurizia Debiaggi (Pavia, Italy) for providing HIV-1PAVIA11 and HIV-1PAVIA12 ; Stefano Buttò (Rome, Italy) for providing HIV-1UG3 ; and Francesca Ceccherini-Silberstein for professional assistance in the sequencing of the viral isolates.
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