Introduction
AIDS has become one of the major diseases of mankind, especially in the Third World, where access to medical care is extremely limited or non-existent. Some of the Third World countries have horrifying rates of HIV-1 and HIV-2 seroprevalence (20–30%), and in the absence of therapeutic interventions, their working populations will be drastically reduced by AIDS in the next few years. Therefore, low-cost medications are badly needed in resource-poor countries. In view of this emergency, the inexpensive anti-malarial agent chloroquine merits consideration. The drug has in-vitro activity against HIV-1 replication and against several AIDS-related opportunistic microorganisms [1,2]. It has a well-documented long-term safety, when dosed for antimalarial prophylaxis and in the treatment of rheumatic diseases. Although no information is available on the in-vivo effects of chloroquine on viral load, its hydroxy analogue hydroxychloroquine has proved in-vivo anti-HIV-1 activity [3,4]. Furthermore, the chloroquine analogue did not impair immune function in immunocompromised patients [4]. In the present study, chloroquine was studied instead of hydroxychloroquine, because of its greater availability in resource-poor countries.
Chloroquine may have effects on several targets in the HIV-1 life cycle. These effects include inhibition of the HIV-1 integrase, inhibition of Tat-mediated transactivation, and a reduction of iron stores within cells affecting reverse transcription [5–7]. Other investigators have demonstrated that chloroquine inhibits HIV-1 replication by affecting the post-transcriptional maturation of gp120 [1], which would make chloroquine an interesting option for combination therapies [7,8]. In this study, we further investigated the mechanism of action of chloroquine, confirming that the drug prevents the maturation of gp120 at concentrations that are clinically relevant and achievable. We have also demonstrated that chloroquine has broad anti-HIV activity, not only against laboratory strains from clade B but also against non-B HIV-1 isolates and against HIV-2.
Materials and methods
Cell cultures
Cells were grown in RPMI 1640 (Gibco Life Technologies, Gaithersburg, MD, USA), supplemented with 10% (v/v) fetal calf serum (Gibco), 200 μg/ml glutamine (Merck, Darmstadt, Germany) and 40 μg/ml gentamicin (Schering-Plough, Milan, Italy), and maintained at 37°C in a humidified atmosphere of 5% carbon dioxide (v/v in air).
The CD4 CXCR4+ MT-4 T cell line was used to assess the inhibitory activity of chloroquine on X4 and R5/X4 strains [9,10]. The persistently HIV-1-infected H9 IIIB cell line was used to test the effects of chloroquine on the post-integrational steps of HIV-1 replication [10]. CD4 CXCR4+ MT-2 cells were used for the titration of X4 strains and in syncytium assays. The CD4 CXCR4+ CEMx174 cell line, kindly provided by Dr P. Gupta, Pittsburgh, PA, USA, was also used in the syncytium assays.
Peripheral blood mononuclear cells (PBMC) were Ficoll-separated, resuspended at a concentration of 106/ml and stimulated for 3 days with 2 μg/ml phytohaemoagglutinin (Difco Laboratories, Detroit, MI, USA).
Viruses
We used the laboratory-adapted HIV-1 IIIB and HIV-2 CBL-20 strains [11], primary isolates from the HIV-1 clades A, B, C, D, E and from the HIV-2 subtypes A and B, and the pRRL.sin.hPGK.GFP reporter construct [12].
Viral stocks were titrated immunoenzymatically using commercially available p24 antigen enzyme-linked immunosorbent assay (ELISA) kits (NEN, Boston, MA, USA) and biologically by the 50% endpoint dilution method, using MT-2 cells (X4 strains) or phytohaemoagglutinin-activated PBMC (R5 isolates). The infectious titre was expressed as tissue culture infecting doses (TCID50)/ml.
Infection of cells with HIV-1 and HIV-2
MT-4 cells or PBMC were infected with viral suspensions at a multiplicity of infection (MOI) of approximately 0.1, as previously described [13]. Cells were then washed three times in phosphate buffered saline, and suspended at 5 × 105/ml in fresh culture medium in the presence or absence of chloroquine (0–12.5 μM) (Sigma, St Louis, MO, USA). In parallel, mock-infected cells were incubated with 0–250 μM chloroquine in order to determine toxicity. HIV-1 p24 was measured by ELISA (NEN and Coulter, Hialeah, FL, USA) in cell culture supernatants at 4 days post-infection (in MT-4 cells) and at 7 days (in PBMC). HIV-2 replication was estimated using SIV p27 ELISA kits (Coulter), which are also able to detect HIV-2 p27 [14]. At different intervals post-infection, cell viability was measured by trypan blue exclusion and by the methyl-tetrazolium (MTT) method [15]. The p24 concentrations and the cell viability values of the uninfected controls on the same day were subjected to linear or non-linear regression and used to calculate the EC50 and IC50 values. Data were normalized using an appropriate transformation when necessary. In the case of non-linear regression, the R2 value was used as a measure of goodness of fit (significance:R2 ≥ 0.95). The selectivity index was calculated as the IC50 : EC50 ratio. In the case of infection of PBMC, a toxicity curve was performed for each donor so as to have a precise estimate of the selectivity index.
In the case of the pRRL.sin.hPGK.GFP, MT-4 cells were infected with the lentiviral construct in the presence or absence of 12.5 μM chloroquine. After 4 days, green fluorescent protein (GFP) expression was detected by flow-cytometry. Mock-transduced cells incubated in the presence or absence of 12.5 μM chloroquine served as the negative controls.
Assays for evaluation of toxicity
In the uninfected controls, cell viability and apoptosis were analysed by trypan blue exclusion, by the MTT method and by propidium iodide/annexin V fluorescein isothiocyanate staining as determined by techniques previously validated in our laboratories [9,15,16].
Assay for viral infectivity
The H9 IIIB cells from a 24 h old culture were washed three times, resuspended in fresh culture medium at 1 × 105 cells/ml and treated for 72 h with 3.12 μM chloroquine. Supernatants of chloroquine-treated and untreated H9 IIIB cells were harvested, tested for HIV-1 p24, and diluted to obtain viral suspensions containing 2 ng of p24/ml (`chloroquine virus’ and ‘control virus', respectively). MT-4 cells were then inoculated with these viral suspensions, washed and resuspended in fresh culture medium. In both the chloroquine virus-infected and control virus-infected cells, one portion was kept untreated and another portion was treated with 3.12 μM chloroquine. At 4 days after viral challenge, p24 was measured in the supernatants.
To evaluate whether chloroquine might alter the capacity of the virus to bind target cells, cell-free virus stocks were overnight incubated at 4°C with 12.5 μg/ml chloroquine or kept under the same conditions in the absence of chloroquine. Uninfected MT-4 cells were then incubated with the viral suspensions for 2 h at 37°C. Cells were then washed, immediately lysed, and subjected to p24 measurement [9].
Syncytium assay
H9 IIIB cells were cultivated in the presence or absence of chloroquine for 48 h, washed three times and then resuspended at 5 × 105/ml. Then, 5 × 104 chloroquine-treated or untreated H9 IIIB cells (100 μl) and 5 × 105 MT-2 (or CEMx174) cells (1 ml) were co-cultivated at 37°C for 6 h. The culture samples were examined under a microscope after 6 h of incubation.
Internal labelling with [35S] cysteine and [35S] methionine and immunoprecipitation
Actively growing H9 IIIB cells were resuspended at a concentration of 105/ml in fresh culture medium and then incubated in the presence or absence of 6.25 μM chloroquine for 72 h. At the end of the incubation period, the cells were washed three times in phosphate buffered saline and resuspended at a density of 107 cells/ml in cysteine- and methionine-free RPMI 1640 (Sigma) plus 10% dialysed fetal calf serum, 1% glutamine and antibiotics. A stock solution of the drug was added to a 2 ml cell suspension. The cells were then metabolically labelled with [35S] cysteine and [35S] methionine (NEN) at 50 μCi/5 × 106 cells for 16 h. After incubation, the viable cells were counted again. The cell suspensions were then centrifuged, and the supernatants were subjected to immunoprecipitation as previously described [16,17], with the different anti-gp120 antibodies, (2G12, F105, antiserum to non-glycosylated HIV-1 gp120) or isotype control antibodies [18–22]. The immunoprecipitated material was then separated by electrophoresis on a 10% polyacrylamide gel and visualized by autoradiography. Autoradiographs were scanned and quantitative analysis was performed with Imagequant Molecular Dynamics software. When necessary, the values were normalized against the numbers of viable cells post-incubation or against the p24 titre in the same supernatants.
Results
Toxicity studies
The first step of this study was to evaluate the toxicity of chloroquine in CD4 CXCR-4+ lymphocytic MT-4 cells. This cell line was chosen as it allows the accurate and reproducible determination of antiviral activity [9,10,13]. On the basis of the toxicity curve, the IC50 of the drug was found to be 47.8 μM. In cells incubated with drug concentrations lower than 20 μM, we observed no significant effects on cell viability, as shown by trypan blue dye exclusion (data not shown), MTT (Fig. 1, panel a) and propidium iodide/annexin staining (not shown). The IC50 of chloroquine in human uninfected PBMC displayed a high level of donor variability (36.1 ± 19.7 μM in seven donors). Cell viability values of PBMC incubated for 7 days with chloroquine concentrations achievable in blood under chronic chloroquine prophylaxis (1–6.25 μM) [23] were similar to those of controls in all donors tested. PBMC cultures grown in the presence of 12.5 μM chloroquine for 7 days displayed a trend to decreased cell viability, compared with control cultures incubated in the absence of chloroquine (median decrease: 25%; first quartile: 1%; third quartile: 37.5%). The difference did not reach statistical significance (Wilcoxon test P = 0.15). To evaluate whether chloroquine might induce apoptosis at the T cell level, primary T cells were cultivated for 10 days in the presence of chloroquine. Annexin V staining indicated that there was no increased apoptosis in T cells incubated with 12.5 μM chloroquine (median fluorescence intensity 133) compared with controls (median fluorescence intensity 136).
;)
Fig. 1.:
Effects of chloroquine on HIV-1 replication. (a) Typical dose-dependent inhibition by chloroquine of HIV-1 IIIB replication in MT-4 cells after 4 days of incubation: calculation of EC50, EC90 and IC50. – – ▿– – Cell viability; ––▴–– HIV-1 p24. (b) Chloroquine decreases the infectivity of newly produced HIV-1. MT-4 cells were infected with HIV-1 IIIB grown in the presence of 3.12 μM chloroquine (chloroquine virus) or with control virus. Of both the chloroquine virus-infected and control virus-infected cells, one portion was kept untreated and another was kept under treatment with 3.12 μM chloroquine. ▪ Control virus; ▓ chloroquine virus; ░ control virus plus chloroquine; □ chloroquine virus plus chloroquine.
Effects of chloroquine on several steps of HIV-1 replication
The MT-4 cells were infected with HIV-1 IIIB and then incubated with chloroquine concentrations achievable in plasma during the prophylaxis and treatment of malaria (1.6, 3.12, 6.25, 12.5 μM) [23]. The drug dose-dependently inhibited p24 production (t-test for regression P < 0.05) with an average EC50 of 3.0 μM, an EC90 of 12.5 μM, and a selectivity index of 15.3 (Fig. 1, panel a). Comparison between the efficacy and toxicity curves confirmed that chloroquine was more potent in inhibiting viral antigen production than cell viability (t-test for slope P < 0.05), as previously reported [7]. Similar results were obtained when the effects of chloroquine on HIV-1 IIIB replication were evaluated in PBMC (EC50 3.1 μM; selectivity index 11.6). The anti-HIV-1 effects of chloroquine were unlikely to be caused by virucidal effects or fusion inhibition, because (i) the drug was added after washing out the virus, and (ii) there was no increased inhibition when the drug was added during the step of virus adsorption onto cells (not shown).
To test whether chloroquine might inhibit reverse transcription and integration within the MT-4 cells, the pRRL.sin.hPGK.GFP reporter construct was used. After entry into target cells, this replication-defective virus is reverse transcribed and integrated into cellular DNA, but carries expression of jellyfish GFP. Expression of this protein allows flow-cytometric detection of the cells containing integrated copies of the GFP gene [12]. The MT-4 cells were infected with pRRL.sin.hPGK.GFP (MOI 0.1, 1, 10) in the presence or absence of 12.5 μM chloroquine. After 48 h, GFP expression was measured by flow-cytometry. Kolmogorov–Smirnov statistics showed no significant differences (P > 0.05) in GFP expression between fluorescence intensity histograms of chloroquine-treated cells and untreated controls, regardless of the MOI used to infect the cells.
To test whether chloroquine might inhibit p24 production in chronically infected cells containing copies of constitutively integrated HIV-1 proviral DNA, H9 IIIB cells were incubated with 3.12–12.5 μM chloroquine. After 72 h, HIV-1 p24 was measured in supernatants. We found that chloroquine had no significant inhibitory effects at any of the concentrations tested [analysis of variance (ANOVA) P = 0.47].
We then tested the infectivity of the virus produced by H9 IIIB cells in the presence of 3.12 μM chloroquine (`chloroquine virus') or not (`control virus'). Supernatants with similar p24 concentrations were harvested from control and chloroquine-treated H9 IIIB cells and diluted to a p24 concentration of 2 ng/ml. Of these suspensions, 1 ml was used to infect the MT-4 cells. After 4 days of incubation, infection was measured as the p24 levels in supernatants. We found that the MT-4 cells infected with the virus from chloroquine-treated H9 IIIB cells displayed lower levels of infection than did cells infected with control virus. Indeed, two-way ANOVA showed that p24 production in MT-4 cells was significantly affected by both chloroquine treatment during cultivation of the challenging virus (P < 0.05) and chloroquine treatment after infection (P < 0.05;Fig. 1, panel b). These results support the hypothesis that chloroquine affects the infectivity of newly produced virus.
Effects of chloroquine on viral envelope glycoproteins
To assess whether the decrease in viral infectivity might be attributed to the envelope glycoproteins produced in the presence of chloroquine, a syncytium assay was performed. H9 IIIB cells were incubated for 3 days in the presence of chloroquine (6.3 μM), washed, and then co-cultured for 6 h with MT-2 cells. We found that pretreatment of H9 IIIB cells with chloroquine impaired syncytial formation in the co-cultures. Two-way ANOVA showed that chloroquine significantly affected syncytium formation when the H9 IIIB cells had been pretreated with chloroquine (P < 0.01), but not when chloroquine had been added only during the co-culture period (P = 0.28) (Fig. 2). Similar results were obtained by co-cultivating the chloroquine-treated or untreated H9 IIIB cells with CEMx174 cells. The effect was dose-dependent and was still observable at 24 h of co-culture (not shown). As the process of syncytial formation is exclusively caused by env-glycoprotein-mediated fusion [24], we concluded that chloroquine affects the production of the viral envelope glycoproteins.
Fig. 2.:
Effects of chloroquine on HIV-1 envelope-mediated fusion. MT-2 cells were co-cultivated for 6 h with H9 IIIB cells pretreated with 6.25 μM chloroquine (chloroquine-pretreated) or maintained in the absence of chloroquine (control). Of both the co-cultures, one portion was kept untreated and another was incubated with chloroquine (6.25 μM). Means ± SEM from three independent experiments. □ Assay in the absence of chloroquine; ▒ assay with 6.25 μM chloroquine.
To confirm that the effects of chloroquine occurred only during intracellular synthesis of env glycoproteins, cell-free virus stocks were incubated with 6.25 μM chloroquine and used to infect the MT-4 cells. Chloroquine-treated and untreated virions were similarly adsorbed onto the MT-4 cells. Lysates from cells incubated for 2 h with chloroquine-treated virus contained an average of 284 ± 74 pg of p24/ml, whereas controls displayed 258 ± 82 pg of p24/ml. Also, the production of p24 at 4 days post-infection was unaffected (not shown), indicating that chloroquine treatment of cell-free virus stocks had no effect on infectivity.
As chloroquine has been reported to affect the terminal glycosylation of some proteins in the trans-Golgi apparatus [25], we decided to test the hypothesis that the drug may affect the glycosylation of gp120. To test this hypothesis, we used two antibodies: 2G12, which recognizes a glycosylated epitope on the outer surface of gp120 and F105, an antibody to the CD4-binding site of gp120, whose binding capacity is independent of glycosylation [19,20]. HIV-1-infected cells were metabolically labelled with [35S] cysteine/methionine in the presence or absence of chloroquine, and supernatants were subjected to immunoprecipitation using 2G12 and F105. The amounts of gp120 immunoprecipitated by the 2G12 antibody were significantly lower in supernatants from chloroquine-treated cells compared with those from the untreated controls (matched-group t-test:P < 0.05;Fig. 3). Instead, there were no quantitative differences in the material immunoprecipitated by the F105 antibody (Fig. 3). Similar results were obtained using an antiserum to non-glycosylated gp120 (not shown). Of note is the fact that glycoproteins from chloroquine-treated cells and revealed by antibodies to non-glycosylated epitopes were slightly but consistently reduced in size (Fig. 3, panel a, second lane from left). This result is consistent with the hypothesis that chloroquine may interfere with the glycosylation of gp120.
Fig. 3.:
Effects of chloroquine on gp120 envelope glycoprotein. H9 IIIB cells were metabolically labelled with [35S] cysteine/methionine in the presence or absence of chloroquine, and gp120 in cell culture supernatants was subjected to immunoprecipitation using the anti-gp120 2G12 and F105 antibodies. (a) Autoradiograph showing material immunoprecipitated by the 2G12 and F105 antibodies in supernatants of H9 IIIB cells treated with chloroquine or untreated. (b) Quantification of the S35 signal as analysed by densitometry (means ± SEM from four independent experiments). *P < 0.05 (matched-group t-test); ▪ control; □ chloroquine 6.25 μM.
Inhibitory activity of chloroquine towards different HIV-1 isolates and HIV-2
So far, our results suggest that alterations in the gp120 glycoprotein may be the primary mechanism of the anti-HIV-1 activity of chloroquine. The genetic diversity of the gp120 glycoproteins has thus to be taken into consideration for the knowledge of the anti-HIV activity of chloroquine. On these grounds, we decided to test whether chloroquine might also inhibit the replication of HIV-1 primary isolates from different subtypes and HIV-2.
Our results indicate that the antiviral activity of chloroquine is not restricted to HIV-1 laboratory strains. Indeed, chloroquine inhibited the replication of a panel of HIV-1 isolates from clades A, B, C, D and E. The drug dose-dependently inhibited replication of the primary isolates within the same range of concentrations displaying antiviral activity towards the HIV-1 IIIB strain (Fig. 1 panel a). The antiviral activity of chloroquine did not seem to depend on co-receptor usage. Chloroquine inhibited not only the X4 isolates tested in MT-4 cells but also the R5 isolates from clades A, C and D tested in PBMC (Fig. 4, panel a). We also assessed chloroquine activity against HIV-2 using the CBL-20 strain (X4, Clade A) [11]. Consistent with the results obtained with HIV-1, chloroquine suppressed HIV-2 CBL-20 replication with an EC50 of 3.0 μM and a selectivity index of 16. The anti-HIV-2 activity of chloroquine was not restricted to subtype A, as the drug also inhibited the replication of non-A HIV-2 isolates (Fig. 4, panel b). For all HIV-1 and HIV-2 isolates tested, inhibition was found to be significantly concentration-dependent according to linear or non-linear regression models (significance threshold P < 0.05 in the case of linear regression and R2 ≥ 0.95 in the case of non-linear regression). In the case of X4 HIV-1 and HIV-2 isolates, the inhibition of viral antigen production was strictly followed by the inhibition of viral cytopathogenicity in the MT-4 cells (not shown). Table 1 shows the EC50, and, where available, the EC90 of chloroquine towards a number of isolates from different HIV-1 and HIV-2 subtypes. Of note is the fact that the EC50 and EC90 found in cells infected at a MOI of 0.01 were lower than those found using the standard MOI of 0.1.
Fig. 4.:
Effects of chloroquine on replication of primary HIV-1 isolates and of HIV-2. (a) Typical HIV-1 p24 values in cell cultures (MT-4 or peripheral blood mononuclear cells; PBMC) infected with X4 primary isolates from HIV-1 clades B (UG20, – –○– –) and E (CA10, —▾—), and R5 primary isolates from clades A (UG3, —▴—), C (VI 829, —▪—) and D (UG1, —" VALIGN="top" COLSEP="0">—), in the presence of different concentrations of chloroquine. (b) Typical HIV-2 p27 values in cell cultures infected with the HIV-2 CBL-20 lab strain (– – ▵ – –, subtype A, MT-4) or with HIV-2 primary isolates (VI 1011, —▿—, subtype B, PBMC; and 7312A, —*—, A/B recombinant, MT-4) in the presence of chloroquine. Cells were lentivirally infected and then incubated in the presence of different chloroquine concentrations. Viral antigen concentrations are presented on a log scale, as measured by enzyme-linked immunosorbent assay in cell culture supernatants at 4 days (MT-4 cells) or at 7 days post-infection (PBMC).
Table 1: Inhibitory activity of chloroquine against different HIV-1 and HIV-2 subtypes.
Discussion
The results of this study indicate that chloroquine at clinically achievable concentrations inhibits HIV-1 replication mainly by affecting the production of the viral envelope glycoprotein(s), and that this drug has broad-spectrum activity against X4, R5 and R5/X4 HIV-1 isolates from subtypes A–E and against HIV-2.
Different mechanisms have been invoked to explain the anti-HIV-1 activity of chloroquine. However, some of them may not be relevant to the antiviral effects observed in this study. For example, the drug has been shown to decrease Tat-mediated transactivation of the HIV-1 long-term repeat [6]. This mechanism may not be occurring in our cell line model system, because p24 production is not affected by chloroquine in chronically infected cells in which Tat inhibitors do affect p24 production [26]. Chloroquine has also been demonstrated to inhibit the HIV-1 integrase in vitro in acellular models [5]. The relevance of this observation in HIV-1-infected cells is also uncertain, because the drug appears to have no effect on GFP expression using the reporter construct pRRL.sin.hPGK.GFP. This evidence indicates that chloroquine has no effects on reverse transcriptase or integrase in the MT-4 cell line. Furthermore, chloroquine was recently shown to restrict the pools of bioactive iron [27]. Iron restriction may result in decreased formation of proviral DNA, as a result of inhibition of the iron-dependent enzyme ribonucleotide reductase [7,28]. However, this mechanism is less likely to explain the antiretroviral activity of chloroquine, because again chloroquine does not appear to interfere with the integration of the pRRL.sin.h PGK.GFP reporter construct.
Chloroquine is a weak base that is known to affect acid vesicles, leading to the dysfunction of enzymes necessary for post-translational modifications of proteins [25]. It is well established that weak bases, by increasing the pH of acidic vesicles, disrupt several enzymes and inhibit the post-translational modification of newly synthesized proteins. We have confirmed that chloroquine and hydroxychloroquine increase the pH in acid vesicles in a dose-dependent fashion, and have demonstrated that HIV-1 passaged on chloroquine- and hydroxychloroquine-treated cells was non-infectious [29]. Tsai et al.[1] demonstrated that the production of gp120 is reduced in T cells and virions isolated from HIV-1-infected cells after chloroquine treatment. Pal et al.[30] also demonstrated that monensin, an ionophore that increases endosomal pH, selectively inhibited gp120 production in a T cell line. These alterations may be responsible for the decreased infectivity of HIV-1 grown in the presence of chloroquine. Our results are in line with this hypothesis. Indeed, chloroquine has no inhibitory effects on p24 production in chronically infected cells, but does affect the infectivity of the virus produced by these cells, indicating that the inhibitory effects of chloroquine cannot be explained by the amounts of virus produced within a single round of HIV-1 replication. Moreover, there was a dose-dependent decrease in syncytial formation when H9 IIIB cells were treated with chloroquine and then co-cultured with uninfected MT-2 or CEMx174 cells. Taken together, these observations strongly suggest that chloroquine affects cellular factors that are essential for the production of gp120. Consistent with the evidence that chloroquine inhibits the infectivity of newly produced virus, inhibition was more evident in cells infected with lower MOI. With a lower MOI, a smaller proportion of cells become infected (i.e. when the virus is still unaffected by the drug), allowing the effects of chloroquine on the newly produced virus to become more evident.
Chloroquine was reported to affect the glycosylation of some cellular and viral glycoproteins [1,25]. For example, Tsai et al.[1] reported that chloroquine inhibits sialylation of the REV-A virus envelope gp90 glycoprotein. We agree with the authors that chloroquine may also affect the terminal glycosylation of HIV-1 gp120. In that study, chloroquine affected the formation of the epitope recognized by the 2G12 antibody, whose binding capacity is dependent on N-linked carbohydrates in the C2, C3, V4, and C4 regions of gp120 [19]. Instead, chloroquine did not affect some of the peptidic epitopes of the gp120 glycoprotein, suggesting that the glycidic but not the aminoacidic portion of the gp120 glycoprotein is affected by chloroquine. In agreement with this evidence, the glycoprotein appeared to be slightly smaller in size after treatment of the H9IIIB cells with chloroquine. In further support of this hypothesis, our preliminary data indicate that chloroquine decreases the incorporation of radiolabelled glucosamine in the envelope glycoproteins from both HIV-1- and HIV-2-infected cells (K. Sperber, manuscript in preparation).
Most of our considerations on the mechanisms of HIV inhibition by chloroquine are based on continuous cell line systems, in that these systems allow the exclusion of bias caused by donor variability. We now show that chloroquine also exerts antiviral effects in PBMC, and that these effects are not confined to laboratory strains, but extend to X4, R5, and X4/R5 primary isolates from clades A–E, as well as to HIV-2.
To our knowledge, this is the first report of the inhibitory effects of chloroquine on non-B HIV isolates. This broad-spectrum activity may be related to the inhibitory effects of chloroquine on the formation of the 2G12 epitope on the gp120 molecule. Indeed, the 2G12 antibody potently and broadly neutralizes primary and T cell line-adapted strains of HIV-1 in vitro[19,24]. Furthermore, cyanovirin, another molecule that interferes with the 2G12 epitope, has anti-HIV-1 and anti-HIV-2 activity [31]. It is therefore possible that chloroquine carries out its antiviral activity by a novel mechanism affecting a conserved portion of the gp120 molecule important for infectivity. Therefore, the anti-HIV activity of chloroquine merits further evaluation.
Further investigation of the anti-HIV activity of chloroquine is also encouraged by a number of considerations. First, the toxicity of chloroquine has been studied longer than that of the currently available antiretroviral drugs, because the drug has been used in the treatment and prophylaxis of malaria for more than half a century and in rheumatic diseases for the past 50 years. Second, not only is chloroquine widely available, but it is also one of the cheapest drugs having anti-HIV-1 activity. Third, chloroquine offers the advantage of having in-vitro and in-vivo activity towards some AIDS-related opportunistic pathogens [2]. Fourth, chloroquine exerts an inhibitory effect on the synthesis of the pro-inflammatory cytokines TNF-α, IL-1 and IL-6, which in turn have favourable effects on HIV replication [32]. Finally, currently available antiretroviral drugs inhibit HIV-1 reverse transcriptase or HIV-1 protease. The effects of chloroquine on the maturation of the viral envelope glycoproteins inhibit viral replication at another step of the HIV-1 replication cycle. In this regard, chloroquine was shown to have additive in-vitro activity in combination with other low-cost drugs, including zidovudine and the combination of didanosine and hydroxyurea or zidovudine and hydroxyurea [7,8,33]. Considering that the inhibition of gp120 may be the primary mechanism of the anti-HIV activity of chloroquine, it appears that the drug affects host cellular enzymes so that the likelihood of the emergence of resistance during chloroquine therapy would be low. A potential limitation of chloroquine as an anti-HIV agent is the fact that the selectivity index of chloroquine is lower than that of presently available antiretroviral drugs. Inhibitory concentrations of chloroquine/hydroxychloroquine towards HIV are, however, clinically achievable and are known to induce no impairment of the immune function in vivo[4].
Conclusion
In-vitro findings demonstrate that chloroquine inhibits HIV by a novel mechanism, and that it has broad-spectrum anti-HIV-1 and anti-HIV-2 activity. Studies of chloroquine using a non-human primate model for HIV infection may be warranted. Obviously, clinical field trials are needed before conclusions can be drawn on the usefulness of the drug in the treatment of HIV infection in resource-poor countries.
Acknowledgements
The authors would like to thank the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, for providing viruses (96USNG31, 93MW960, CBL-20/H9, 7312A, and CDC310319) and antibodies (2G12, F105 and an antiserum to non-glycosylated gp120), Dr K. Fransen, Institute of Tropical Medicine, Antwerp, Belgium, for providing VI 390, VI 829, VI 1011, VI 1249, VI 1415 and CA 10 primary isolates, Dr S. Buttò, Istituto Superiore di Sanità (ISS), Rome, Italy, for providing the UG1, UG3, UG20 primary isolates, and Dr L. Naldini, IRCC, Candiolo, Italy, for providing the reporter construct pRRL.sin.hPGK.GFP. The authors are also grateful to Dr S. Buttò for critically reading the manuscript. Dr A. Savarino is personally grateful to Dr G.P. Pescarmona, University of Turin, Turin, Italy, for enlightening discussion.
References
1. Tsai WP, Nara PL, Kung HF, Oroszlan S. Inhibition of human immunodeficiency virus infectivity by chloroquine. AIDS Res Hum Retroviruses 1990, 6: 481–489.
2. Boelaert JR, Appelberg R, Gomes MS.
et al. Experimental results on chloroquine and AIDS opportunists. J Acquir Immune Defic Syndr Hum Retrovir 2001, 26: 300–301.
3. Sperber K, Chiang G, Chen H.
et al. Comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with HIV-1. Clin Ther 1997, 19: 913–923.
4. Sperber K, Louie M, Kraus T.
et al. Hydroxychloroquine treatment of patients with human immunodeficiency virus type 1. Clin Ther 1995, 17: 622–636.
5. Fesen MR, Kohn KW, Leteurtre F, Pommier Y. Inhibitors of human immunodeficiency virus integrase. Proc Natl Acad Sci USA 1993, 90: 2399–2403.
6. Jiang MC, Lin JK, Chen SS. Inhibition of HIV-1 Tat transactivation by quinacrine and chloroquine. Biochem Biophys Res Commun 1996, 226: 1–7.
7. Savarino A, Gennero L, Boelaert JR, Sperber K. The anti-HIV-1 activity of chloroquine. J Clin Virol 2001, 20: 131–135.
8. Boelaert JR, Sperber K, Piette J. Chloroquine exerts an additive in vitro anti-HIV type 1 effect when associated with didanosine and hydroxyurea. AIDS Res Hum Retroviruses 1999, 15: 1241–1247.
9. Savarino A, Bottarel F, Calosso L.
et al. Effects of the human CD38 glycoprotein on the early stages of the HIV-1 replication cycle. FASEB J 1999, 13: 2265–2276.
10. De Clercq E. Basic approaches to anti-retroviral treatment. J Acquir Immune Defic Syndr 1991, 4: 207–218.
11. Berry N, Ariyoshi K, Balfe P, Tedder R, Whittle H. Sequence specificity of the human immunodeficiency virus type 2 (HIV-2) long terminal repeat u3 region in vivo allows subtyping of the principal HIV-2 viral subtypes A and B. AIDS Res Hum Retroviruses 2001, 17: 263–267.
12. Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998, 72: 9873–9880.
13. Savarino A, Pugliese A, Martini C, Pich PG, Pescarmona GP, Malavasi F. Investigation of the potential role of membrane CD38 in protection against cell death induced by HIV-1. J Biol Reg Homeost Agents 1996, 10: 13–18.
14. Lairmore MD, Hofheinz DE, Letvin NL, Stoner CS, Pearlman S, Toedter GP. Detection of simian immunodeficiency virus and human immunodeficiency virus type 2 capsid antigens by a monoclonal antibody-based antigen capture assay. AIDS Res Hum Retroviruses 1993, 9: 565–571.
15. Savarino A, Calosso L, Piragino A.
et al. Modulation of surface transferrin receptors in lymphoid cells de novo infected with human immunodeficiency virus type 1. Cell Biochem Funct 1999, 17: 47–55.
16. Chen H, Yip YK, George I, Tyorkin M, Salik E, Sperber K. Chronically HIV-1 infected monocytic cells induce apoptosis in co-cultured T cells. J Immunol 1998, 161: 4257–4267.
17. Sperber K, Shaked A, Posnett DN, Hirschman SZ, Bekesi JG, Mayer L. Surface expression of CD4 does not predict susceptibility to infection with HIV-1 in human monocyte hybridomas. J Clin Lab Immunol 1990, 31: 151–158.
18. Buchager A, Predl R, Strutzenberger K.
et al. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein–Barr virus transformation for peripheral blood lymphocyte immunization. AIDS Res Hum Retroviruses 1994, 10: 359–369.
19. Trkola A, Purtscher M, Muster T.
et al. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J Virol 1996, 70: 1100–1108.
20. Posner MR, Cavacini LA, Emes CL, Power J, Byrn R. Neutralization of HIV-1 by F105, a human monoclonal antibody to the CD4 binding site of gp120. J Acquir Immune Defic Syndr 1993, 6: 7–14.
21. Posner MR, Hideshima T, Cannon T, Mukherjee M, Mayer KH, Byrn RA. An IgG human monoclonal antibody that reacts with HIV-1 gp120, inhibits virus binding to cells, and neutralizes infection. J Immunol 1991, 146: 4325–4332.
22. Haigwood NL, Schuster JR, Moore GK.
et al. Importance of hypervariable regions of HIV-1 gp120 in generation of virus neutralizing antibodies. AIDS Res Hum Retroviruses 1990, 6: 855–869.
23. Ducharme J, Farinotti R.
Clinical pharmacokinetics and metabolism of chloroquine.Focus on recent advancements. Clin Pharmacokinet 1996, 31: 257–274.
24. Parren PWHI, Moore JP, Burton DR, Sattenau Q. The neutralizing antibody response to HIV-1: viral evasion and escape from humoral immunity. AIDS 1999, 13 (Suppl. A) : S137–S162.
25. Thorens B, Vassalli P. Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion. Nature 1986, 321: 618–620.
26. Shoji S, Furuishi K, Misumi S, Miyazaki T, Kino M, Yamataka K. Thiamine disulfide as a potent inhibitor of human immunodeficiency virus (type-1) production. Biochem Biophys Res Commun 1994, 205: 967–975.
27. Morra E, Savarino A, Gennero L, Pescarmona GP. Effects of chloroquine on iron metabolism in a lymphocytic cell line. J Clin Virol 2000, 16: 91–92.
28. Savarino A, Pescarmona GP, Boelaert JR. Iron metabolism and HIV infection: reciprocal interactions with potentially harmful consequences? Cell Biochem Funct 1999, 17: 279–287.
29. Chiang G, Sassaroli M, Louie M, Chen H, Stecher VJ, Sperber K. Inhibition of HIV-1 replication by hydroxychloroquine: mechanism of action and comparison with zidovudine. Clin Ther 1996, 18: 1080–1092.
30. Pal R, Gallo RC, Sarngadharan MG. Processing of the structural proteins of human immunodeficiency virus type 1 in the presence of monensin and cerulenin. Proc Natl Acad Sci USA 1988, 85: 9283–9286.
31. Mori T, Boyd MR. Cyanovirin-N, a potent human immunodeficiency virus-inactivating protein, blocks both CD4-dependent and CD4-independent binding of soluble gp120 (sgp120) to target cells, inhibits sCD4-induced binding of sgp120 to cell-associated CXCR4, and dissociates bound sgp120 from target cells. Antimicrob Agents Chemother 2001, 45: 664–672.
32. Boelaert JR, Piette J, Sperber K. The potential place of chloroquine in the treatment of HIV-1-infected patients. J Clin Virol 2001, 20: 137–140.
33. Boelaert JR, Sperber K, Piette J. Chloroquine exerts an additive in vitro anti-HIV-1 effect, when combined to zidovudine and hydroxyurea. Biochem Pharmacol 2001, 61: 1531–1535.
