Human Immunodeficiency Virus Type 1 Escape ... - Journal of Virology

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Mar 30, 2009 - Xiang, S. H., P. D. Kwong, R. Gupta, C. D. Rizzuto, D. J. Casper, R. Wyatt,. L. Wang, W. A. Hendrickson, M. L. Doyle, and J. Sodroski. 2002.
JOURNAL OF VIROLOGY, Sept. 2009, p. 9577–9583 0022-538X/09/$08.00⫹0 doi:10.1128/JVI.00648-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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NOTES Human Immunodeficiency Virus Type 1 Escape from Cyclotriazadisulfonamide-Induced CD4-Targeted Entry Inhibition Is Associated with Increased Neutralizing Antibody Susceptibility䌤 Kurt Vermeire,1* Kristel Van Laethem,1 Wouter Janssens,2 Thomas W. Bell,3 and Dominique Schols1 Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium1; Institute of Tropical Medicine, Antwerp, Belgium2; and Department of Chemistry, University of Nevada, Reno, Nevada3 Received 30 March 2009/Accepted 21 June 2009

Continuous specific downmodulation of CD4 receptor expression in T lymphocytes by the small molecule cyclotriazadisulfonamide (CADA) selected for the CADA-resistant human immunodeficiency virus type 1 (HIV-1) NL4.3 virus containing unique mutations in the C4 and V5 regions of gp120, likely stabilizing the CD4-binding conformation. The amino acid changes in Env were associated with decreased susceptibility to anti-CD4 monoclonal antibody treatment of the cells and with higher susceptibility of the virus to soluble CD4. In addition, the acquired ability of a CADA-resistant virus to infect cells with low CD4 expression was associated with an increased susceptibility of the virus to neutralizing antibodies from sera of several HIV1-infected patients.

Treatment of T cells with CADA resulted in a time-dependent reduction in cell surface CD4 expression (88% decrease, as shown by staining with anti-CD4 MAbs) (Fig. 1A). These results were compared with results from treatment of the cells with RPA-T4, a MAb that binds to the extracellular NH2terminal D1 region of CD4. RPA-T4 MAb treatment also resulted in a time-dependent decrease in the number of CD4 molecules at the cell surface (Fig. 1A). Flow cytometric staining for CD4 (with allophycocyanin [APC]-labeled SK3 antibody) showed a 60% CD4 downmodulation induced by RPA-T4 treatment, an observation that is in line with the receptor-downmodulating activity of other CD4-binding MAbs (12, 28). A major challenge in the treatment of HIV is the emergence of escape mutants that are no longer susceptible to drugs (27). We therefore investigated the profile of HIV type 1 (HIV-1) in vitro resistance to this viral entry inhibitor. By culturing the HIV-1 CXCR4-tropic (X4) molecular clone NL4.3 (HIV1NL4.3) in MT-4 cells in increasing concentrations of CADA, we selected for a virus that showed reduced susceptibility to the compound (Fig. 1B). The virus that was isolated after 40 passages in cell culture became insensitive to the CD4 receptor-downmodulating effect of CADA (CADAres40) (Table 1). Infection of MT-4 cells by the escape mutant was inhibited by different reverse transcriptase, protease, and entry inhibitors in a manner similar to that for the wild-type (WT) counterpart subcultivated in parallel in control medium (WT40), as evidenced by the comparable 50% effective concentrations (EC50s) (Table 1). However, for the anti-CD4 MAb RPA-T4, a significant decrease in activity against the CADA-resistant virus compared to that against the WT virus was noted (sixfold

The introduction of highly active antiretroviral therapy has revolutionized the management of human immunodeficiency virus (HIV) infections. Recently, the CCR5 receptor antagonist maraviroc was approved by the FDA for antiretroviral therapy of HIV and thus became the first approved drug that inhibits HIV by targeting a cellular receptor. In addition, clinical studies with the anti-CD4 monoclonal antibody (MAb) ibalizumab (formerly TNX-355) demonstrated antiviral activity in vivo and proved the feasibility of targeting the CD4 receptor without CD4⫹ T-cell depletion or serious adverse effects (20). Cyclotriazadisulfonamide (CADA) compounds represent a new class of small-molecule HIV entry inhibitors (40). We previously demonstrated that the lead compound CADA specifically decreases surface CD4 in several types of human CD4⫹ cells (i.e., T lymphocytes, monocytes, and dendritic cells), producing broad-spectrum antiviral potency against infection by laboratory HIV strains and clinical isolates belonging to various clades, independent of tropism (37–39, 41). The compound does not directly interact with gp120 or cell surface CD4; it specifically decreases cellular biosynthesis of CD4 (unpublished data). CADA compounds are small-molecule alternatives to therapeutic strategies involving CD4 gene knockdown (29), CD4-specific designed ankyrin repeat proteins (34), and CD4-binding/downmodulating MAbs (20, 28). CADA was synthesized as described in detail elsewhere (5). * Corresponding author. Mailing address: Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. Phone: 32-16-337341. Fax: 32-16-337340. E-mail: [email protected]. 䌤 Published ahead of print on 1 July 2009. 9577

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FIG. 1. (A) Time-dependent CD4 receptor downmodulation by CADA and anti-CD4 MAb RPA-T4. MT-4 cells were treated with CADA (10 ␮g/ml) or the unlabeled anti-CD4 MAb RPA-T4 (10 ␮g/ml) for 30 min with cooling on ice (“30 min; 0°C”) or at 37°C for 4 h (“4 h; 37°C”), 24 h (“1 d; 37°C”), or 72 h (“3 d; 37°C”). At the different time points, cells were washed and stained for CD4 expression. Cell surface CD4 staining was performed by simultaneous administration of two anti-CD4 MAbs recognizing distinct epitopes of the receptor, phycoerythrin (PE)-labeled anti-CD4 MAb clone RPA-T4 (eBioscience, San Diego, CA) and APC-labeled anti-CD4 MAb clone SK3 (BD Biosciences, San Jose, CA). By this

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TABLE 1. Profiles of WT40 and CADAres40 HIV-1NL4.3 virus susceptibility to antiviral agentsa EC50b

Agent WT40

CADAres40

CADA (␮g/ml) 1.2 ⫾ 0.2 ⬎50 Zidovudine (nM) 11.0 ⫾ 1.7 6.4 ⫾ 1.4 Tenofovir (␮M) 4.1 ⫾ 0.9 3.2 ⫾ 0.8 Delavirdine (ng/ml) 18.5 ⫾ 1.7 11.4 ⫾ 2.9 Efavirenz (ng/ml) 1.1 ⫾ 0.2 1.0 ⫾ 0.3 Saquinavir (ng/ml) 13.2 ⫾ 3.9 8.5 ⫾ 3.1 Indinavir (ng/ml) 23.8 ⫾ 5.9 12.5 ⫾ 2.5 Lectin GNAe 140.8 ⫾ 57.7 100.8 ⫾ 51.4 (ng/ml) f Lectin HHA 148.4 ⫾ 60.8 106.0 ⫾ 43.7 (ng/ml) Enfuvirtide (ng/ml) 56.0 ⫾ 10.8 62.3 ⫾ 10.2 AMD3100 (ng/ml) 7.4 ⫾ 2.1 9.4 ⫾ 2.3 DS-5000 (␮g/ml) 0.81 ⫾ 0.18 0.70 ⫾ 0.23 Anti-CD4 MAb 0.32 ⫾ 0.08 1.97 ⫾ 0.51 RPA-T4 (␮g/ml)

Fold Fold resistancec sensitivityd

⬎42 2 1 2 1 2 2 1 1 1 1 1 6

a WT NL4.3 virus was selected after 40 passages in control medium (WT40), and CADA-resistant virus was selected after 40 passages in CADA compound (CADAres40), as described in the legend for Fig. 1B. The antiviral activities of the compounds were tested using MT-4 cells infected with equal inputs of these WT40 and CADAres40 viruses (50% tissue culture infective doses of 50). After 5 days of infection, the supernatant was collected and tested for HIV-1 p24 antigen content by enzyme-linked immunosorbent assay. b Values are means ⫾ standard errors of the means from three to five different experiments. The units of measure are given in the first column. c Resistance was calculated as follows: (EC50 CADAres40)/(EC50 WT40). Values shown in boldface type indicate significant differences as given in the text. d Sensitivity was calculated as follows: (EC50 WT40)/(EC50 CADAres40). e GNA, Galanthus nivalis agglutinin. f HHA, Hippeastrum sp. hybrid agglutinin.

increase in EC50; P ⫽ 0.015 by Student’s t test). The partial cross-resistance and reduced susceptibility of the CADA-resistant virus to this MAb (Fig. 2B) are consistent with the moderate CD4-downmodulating activity of RPA-T4 and the ability of the CADA-resistant virus to utilize lower levels of CD4. Analysis of the genome of the CADA-resistant virus revealed only five amino acid mutations in the envelope glycoprotein gp120: Y7H, V68A, Q308H, S438R, and S463P (Fig. 1C, CA40). After 20 passages of CADA selection, we observed two substitutions in the viral gp120: V68A and S438R (Fig. 1C,

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CA20). However, these two mutations were responsible for only a fourfold decrease in sensitivity to CADA. After 20 additional passages, three extra mutations occurred: Y7H, Q308H, and S463P. The mutations Y7H and V68A lie within the N-terminal region of gp120, which is not involved in CD4 binding (23). Substitution Q308H in the hypervariable loop V3 of the CADA-resistant virus seems to occur frequently in other in vitro-generated resistant viruses: we noted this mutation in NL4.3 virus resistant to the CXCR4 antagonist AMD3100 (7), SDF-1 (33), and the broadly neutralizing MAb 2G12 (19). Substitution S438R (C4 region of gp120) has been observed in some viruses that are resistant to BMS-378806 (corresponding mutation, S440R) (13, 25), a small-molecule compound that inhibits CD4 attachment by binding to gp120 and altering its conformation (16). Residue 438 lies outside both the CD4 and the BMS-378806 binding site (6), so the S438R mutation may help to stabilize the CD4-binding conformation of gp120. This substitution is apparently responsible for the modest (fourfold) resistance of the 20-passage mutant, which also contains the irrelevant V68A mutation (Fig. 1C, CA20). Substitutions Q308H and S463P apparently confer most of the (70-fold) CADA resistance of the 40-passage mutant (Tables 1 and 2). The serine residue at position 463 (corresponding to 465 in the crystal structure of CD4-liganded gp120) (23) lies in the V5 loop linking two CD4-binding sequences (453 to 457 and 467 to 472). Proline is unique in its ability to restrict protein conformations, so the S463P mutation may further enhance CD4 binding by favoring the liganded conformation of gp120. This conformational mechanism explains the observed cross-resistance toward BMS-378806, which does not bind to CD4-liganded gp120 (16). The S463P mutation also affects an N-glycosylation site sequon, resulting in the loss of a potential N-glycosylation site (Fig. 1C). Deletion of N glycosylations in gp120 has been reported to occur during development of HIV-1 resistance to other entry inhibitors, such as MAb 2G12 (19), the mannose-specific plant lectins (3), and several other carbohydrate-binding agents (1, 2, 4). In contrast, deletion of N glycosylation in gp120 sometimes results in an enhanced neutralizing capacity of antiviral agents, e.g., carbohydrate-binding agents, against 2G12-resistant virus (19). In

means, we could discriminate between competition for receptor binding (through steric hindrance) and downregulation of the complete receptor. The CADA compound does not bind cell surface CD4 but downmodulates the expression of the CD4 receptor, as shown by the similar effects of both anti-CD4 MAbs. The CD4-downmodulating effect of CADA was confirmed with a panel of six other anti-CD4 MAbs, all showing the removal of the CD4 receptors from the cell surface after 24 h of treatment (not shown). RPA-T4 antibody treatment of the cells resulted in a steric hindrance for PE-labeled RPA-T4 staining (detectable after 30 min treatment at 0°C) but also in more than 50% CD4 downregulation after 3 days of treatment (determined with APC-labeled SK3). CD4 MAb binding is given as the percentage of stained nontreated control cells (based on the mean fluorescence intensity values). Comparable results were also obtained with peripheral blood mononuclear cells (data not shown). The bars represent the means ⫾ standard errors of the means from three independent experiments. (B) Selection of CADA-resistant virus in MT-4 cells. Cells and HIV-1 X4NL4.3 virus were subcultivated in the presence of CADA every 4 days and microscopically examined for signs of virus replication. Following virus breakthrough, the supernatant was collected and stored and used to infect fresh cell cultures with a higher concentration of CADA. The procedure was repeated up to a concentration of 50 ␮g/ml of CADA (i.e., 40 passages; CADAres40; red line). In parallel, cells were infected with WT HIV-1 X4NL4.3 virus (WT1) and subcultivated in control medium without compound for 40 passages (WT40; blue dashed line). (C) gp120 amino acid sequences of WT and CADA-resistant viruses. HIV-1 X4NL4.3 virus was selected as described for panel B. At passages 1, 20, and 40, the collected supernatant was subjected to genome sequencing. The figure shows the amino acid sequences of the original virus (WT01; black), the WT control virus after 40 passages in medium without compound (WT40; blue), and the CADA-resistant virus after 20 and 40 passages in medium with CADA (CA20 and CA40, respectively; red). The variable regions are indicated as V1 to V5. The residues of gp120 involved in CD4 binding (according to reference 23) are starred. The numbering of the amino acids is in comparison to a reference strain (GenBank accession no. AF324493). Five mutations in the gp120 genome of CADA-resistant virus were observed (Y7H, V68A, Q308H, S438R, and S463P), with the loss of the potential N-glycosylation site at position 461 in the V5 region.

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FIG. 2. (A) Enhanced anti-gp120 antibody binding to CADA-resistant virus. MT-4 cells were infected with CADAres40 and WT40 viruses. After 3 days, when comparable levels of infection were observed for WT virus- and CADA-resistant virus-infected cell cultures, cells were collected, washed, stained for gp120 with the V3-recognizing antibodies (Abs) 4030 and 1511 and the CD4 binding site-recognizing antibody b12, and detected with a phycoerythrin-labeled goat anti-human antibody. Ab 4030 neutralizes virus that contains the GPGR sequence at the apex of the V3 loop. Ab 1511 reacts with V3 epitope HIGPGR; mutation Q308H modifies QRGPGR (WT; no binding) into HRGPGR (CADA resistant; binding). The amount of Ab binding is expressed as the mean fluorescence intensity (MFI). The results are representative of three to six independent experiments. (B) Neutralization susceptibility of CADA-resistant virus. MT-4 cells were infected with CADAres40 and WT40 viruses in the presence of fivefold dilutions of the gp120-recognizing antibodies 4030, 2G12, and b12; the anti-CD4 MAb RPA-T4; the small-molecule gp120 binding agent BMS-378806; and sCD4. After 5 days, when untreated cells were strongly infected, supernatant was collected and HIV-1 p24 antigen content was determined. For each concentration of the antiviral agent, the degree of neutralization was calculated by comparison to that for the untreated infected cells. The means ⫾ standard errors of the means from three independent experiments are shown. (C) Susceptibility of CADA-resistant virus to neutralizing sera. A panel of seven HIV-1-positive plasma samples were evaluated for their capacity to neutralize CADAres40 and WT40 viruses. The viruses were selected as described in the legend for Fig. 1B. Next, a dilution range of virus was incubated with the serum samples for 1 h and subsequently administered to phytohemagglutinin-stimulated peripheral blood mononuclear cells. After 2 h of adsorption, cells were washed and incubated for 14 days. The amount of infection was measured by HIV p24 antigen-specific enzyme-linked immunosorbent assay, and neutralization indices and the corresponding percentages of neutralization were calculated as described in reference 9.

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TABLE 2. Profiles of recombinant pNL4.3 env WT40 and recombinant env CADAres40 virus susceptibility to antiviral agentsa EC50b

Agent WT40

CADA (␮g/ml) 0.7 ⫾ 0.1 Tenofovir (␮M) 1.9 ⫾ 0.9 Lopinavir (ng/ml) 16.3 ⫾ 2.8 Raltegravir (nM) 3.9 ⫾ 0.4 Enfuvirtide (ng/ml) 59.0 ⫾ 18.8 AMD3100 (ng/ml) 2.5 ⫾ 0.2 Maraviroc (ng/ml) ⬎1,000 Anti-CD4 MAb 0.36 ⫾ 0.08 RPA-T4 (␮g/ml) Anti-gp120 MAb 109.1 ⫾ 15.6 b12 (ng/ml) sCD4 (ng/ml) 120.5 ⫾ 37.0 CD4M33 (ng/ml) 45.3 ⫾ 18.6

CADAres40

⬎50 1.3 ⫾ 0.4 8.0 ⫾ 0.8 2.0 ⫾ 1.1 36.7 ⫾ 11.3 3.0 ⫾ 0.2 ⬎1,000 1.98 ⫾ 0.54

Fold Fold resistancec sensitivityd

⬎71 1 2 2 2 1 6

77.0 ⫾ 24.8

1

28.1 ⫾ 5.2 19.6 ⫾ 11.3

4 2

a Recombination of gp160 sequences of CADAres40 and WT40 viruses into a proviral WT HIV-1 clone (pNL4.3) from which the corresponding env gene was deleted was performed. The amino acid sequences of the recombinants were determined (in comparison to that of pNL4.3 关GenBank accession no. AF324493兴) and confirmed the presence of the CADA resistance mutations (as depicted in Fig. 1C). b The antiviral activities of the compounds were tested using MT-4 cells infected with equal inputs of recombinant pNL4.3 env WT and CADA-resistant NL4.3 viruses (50% tissue culture infective dose of 50). After 5 days of infection, the supernatant was collected and tested for HIV-1 p24 antigen content by enzyme-linked immunosorbent assay. Values are means ⫾ standard errors of the means from three to seven different experiments. The units of measure are given in the first column. c Resistance was calculated as follows: (EC50 CADAres40)/(EC50 WT40). Values shown in boldface type indicate significant differences. d Sensitivity was calculated as follows: (EC50 WT40)/(EC50 CADAres40).

this regard, we observed a slightly higher neutralizing potency of MAb 2G12 against the CADA-resistant virus than against the WT virus (Fig. 2B). However, during in vitro resistance selection N-glycosylation deletions in the viral envelope can easily be tolerated and occur spontaneously. Next, we wanted to verify if the viral envelope of the escape virus was solely responsible for the decreased susceptibility to CADA. Based on chimeric virus technology (11), we performed the recombination of the env gene gp160 sequences of the CADAres40 and WT40 viruses into a proviral WT HIV-1 clone (pNL4.3) from which the corresponding env gene was deleted (11). Both recombinant viruses proved to exert similar infection efficiencies (not shown). When the pNL4.3 WT and CADA-resistant viruses were tested for susceptibility to various anti-HIV agents, we noted for the recombinant viruses a susceptibility profile that was comparable to that of the corresponding native strains. Replication of the pNL4.3 CADAresistant mutant could not be prevented by CADA treatment (up to 50 ␮g/ml), indicating that the viral envelope was solely responsible for CADA resistance (Table 2). Again, the CADAresistant virus proved to be significantly less susceptible than the WT virus to anti-CD4 MAb treatment of the cells (EC50s were 2.0 and 0.4 ␮g/ml, respectively; P ⫽ 0.021, Student’s t test) (Table 2). In contrast, there was a tendency for soluble CD4 (sCD4) (35) to exert a higher neutralizing activity against the CADA-resistant virus (Fig. 2B): we measured a 4.3-fold increase in potency compared to that for the WT virus (P ⫽ 0.056, Student’s t test) (Table 2). The anti-gp120 MAb b12 showed no difference in activity toward the CADA-resistant

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virus versus the WT virus (Fig. 2A and B; Table 2). This is consistent with reports that so-called CD4 binding site antibodies, such as b12, recognize conformations of gp120 different from that recognized by CD4 and CD4-induced antibodies, which are induced by the CD4-liganded conformation of gp120 (42). Finally, we further explored the neutralizing susceptibility of the CADA escape mutant CADAres40 and found that several anti-gp120 MAbs bind to the viral envelope of CADA-resistant virus more efficiently than to that of WT virus (Fig. 2A). In addition, by means of neutralizing antibodies isolated from HIV-1-infected individuals, as described elsewhere (9), we observed that the CADA-resistant virus was markedly more prone than the WT to the neutralizing capacities of the different sera (Fig. 2C). Whereas the WT virus could be neutralized by only half of the serum samples, the CADAresistant virus proved to be susceptible to virtually all of the tested sera (Fig. 2C). Conformational flexibility and N glycosylation of gp120 are believed to be two important strategies used by HIV to evade the immune system (8, 24). The observed mutations in CADAresistant gp120 apparently stabilize the conformation of gp120 and reduce glycosylation, both of which can enhance the ability to elicit neutralizing antibodies (8, 24). Increased susceptibility to neutralizing antibodies has also been reported for CD4independent viruses (17), which could explain the rarity of strict CD4-independent HIV variants in vivo (10, 22). However, these viruses still show higher infectivity and replication ability when CD4 is expressed on the cell surface. Here, CADA resistance is not associated with escape of CD4 receptor usage, as the resistant virus was not able to infect CD4-negative U87.CXCR4⫹ cells or other CD4-negative cells, such as the SupT1 clone B2 (not shown). A similar loyalty of HIV-1 to its receptor has also been reported for R5 viruses resistant to CCR5 antagonists and X4 escape mutants to CXCR4 inhibitors (26, 31, 33, 36). In the HIV entry process, multiple CD4 and coreceptor binding events are needed to efficiently activate a viral spike, consisting of three gp120 molecules noncovalently attached to three subunits of gp41, thus implying that CD4 receptor and coreceptor densities have a crucial role in the efficiency of HIV infection. Accordingly, several studies have already demonstrated that (co)receptor density levels in individuals are rate limiting for infection (21, 30) and that downregulation of CCR5 expression, e.g., by the G1 cytostatic agent rapamycin, can be an interesting strategy for both the prevention and the treatment of HIV infection (14, 15). In addition, not only could inhibiting the expression of a cellular receptor have a direct antiviral effect by preventing viral entry, but it is possible that this could force the virus to become less dependent on use of its receptor by changing its viral envelope. Hypothetically, these escape mutants could then be eliminated more efficiently by neutralizing antibodies, so that in this case development of resistance to the drug might even be considered favorable, providing a new way of trapping the virus. Recently, and in line with our results, it has been reported that sequence changes in the envelope that confer resistance to other HIV entry inhibitors can increase the accessibility of some neutralizing antibody epitopes and ultimately make these viruses more prone to antibody neutralization (18, 31, 32).

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In conclusion, CADA-resistant virus selected for mutations in the viral envelope that support the infection of cells with low CD4 expression. The amino acid substitutions were associated with decreased susceptibility to anti-CD4 MAb treatment and with higher sensitivity to neutralizing antibodies from HIVinfected patients. These in vitro observations may have interesting implications for novel antiretroviral therapies and can open new roads to the successful design of an HIV vaccine. HIV-1 V3 MAbs 447-52D (4030) and 268-D IV (1511) were obtained through the AIDS Research and Reference Program, Division of AIDS, NIAID, NIH, from Susan Zolla-Pazner. The human immunoglobulin G1 b12 antibody and the CD4 mimic CD4M33 were kind gifts from Dennis R. Burton and Loïc Martin, respectively. We thank Sandra Claes, Eric Fonteyn, Becky Provinciael, and Yoeri Schrooten for excellent technical assistance. We are grateful to Kris Covens for helping with the pNL4.3 recombinant viruses and to Betty Willems for assisting with the neutralization experiments. Kurt Vermeire is a postdoctoral researcher at the Fonds voor Wetenschappelijk Onderzoek (F.W.O.)—Vlaanderen (Belgium). This work was supported by the Centers of Excellence from the Katholieke Universiteit Leuven (EF/05/15), EMPRO 503558, Fondation Dormeur, and F.W.O. (G-485-08).

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