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Institut National de la Santé et de la Recherche Médicale Unité II 430 and Université Pierre et Marie Curie, Hôpital Broussais, Paris, France;
Centre Integré de Recherches Biocliniques sur le SIDA (CIRBS), Hôpital St. Joseph, Paris, France;
Institut Pasteur, Paris, France;
Fondazione Centro S. Raffaele Del Monte Tabor, Milan, Italy; and
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Consiglio Nazionale delle Ricerche (CNR) Laboratory, Milan, Italy.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CXCR4 and CCR5 chemokine receptors function as the major coreceptors for T lymphocyte and macrophage-tropic HIV-1 isolates, also referred to as X4 and R5 isolates, respectively (5, 6). R5-tropic strains account for most sexually transmitted HIV infections (7). The importance of CCR5 for HIV-1 transmission was evidenced by the observation that individuals homozygous for a defective CCR5 allele remained uninfected despite repeated exposure to HIV (8, 9, 10, 11, 12). The CCR5 molecule is thus being considered as a target for therapeutic strategies designed to block HIV-1 entry into cells (13).
In the present study, we demonstrate that i.v. Ig contains Abs that recognize a CCR5 peptide motif corresponding to the N-terminus of CCR5 (II.E/C-CCR5) molecule. Affinity-purified anti-CCR5 Abs bound to the CCR5 molecule expressed on transfected Chinese hamster ovary (CHO) cells and on monocyte-derived macrophages (MDM) and blocked the binding of RANTES to its receptor. Anti-CCR5 Ig further inhibited the infection of monocytes/macrophages and lymphocytes by both primary and laboratory-adapted R5-tropic HIV-1 strains.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intravenous Ig (Sandoglobulin) was a gift from the Central Laboratory of the Swiss Red Cross (Bern, Switzerland). F(ab')2 were prepared from i.v. Ig by pepsin digestion (2% w/w; Sigma, St. Louis, MO) in acetate buffer, pH 4.1, for 18 h at 37°C, followed by chromatography on protein G-Sepharose. F(ab')2 were free of intact IgG and Fc fragments, as assessed by SDS-PAGE and ELISA. FITC-conjugated mAb to CD4 (Leu-3a) was purchased from BD Biosciences (Le Pont de Claix, France). PE-conjugated mAb to CCR5 (clone 2D7) and PE-conjugated mAb to human RANTES (clone 2D5) were obtained from PharMingen (Le Pont de Claix, France). The PE cyanin5-conjugated anti-CD14 (RMO52) was obtained from Immunotech Beckman Coulter (Villepinte, France). Biotinylated goat anti-mouse IgG and PE-conjugated goat anti-human IgG were obtained from Jackson ImmunoResearch (Immunotech, Marseille, France). M-CSF and the chemokines RANTES, SDF-1, and IL-2 were obtained from R&D Systems Europe (Abingdon, Oxon, U.K.). PHA was obtained from Sigma, and the CPRG reagent was obtained from Boehringer Mannheim (Meylan, France).
The peptide CCR5 (MDYQVSSPIYDINYYTSEPC) that corresponds to the N-terminus of CCR5 was synthesized by the solid-phase F-moc method using an Applied Biosystems model 433A peptide synthesizer (Foster City, CA) (14).
Immunoaffinity purification of anti-CCR5 Abs
For affinity purification of anti-CCR5 Ig, the CCR5 peptide (20 aa) was coupled to activated CH Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden), and the column was equilibrated with PBS, pH 7, before being loaded with 50 mg of i.v. Ig per 2 ml of matrix containing 3 mg of the peptide. i.v. Ig was allowed to interact with the matrix overnight at 4°C before washing the column with PBS, pH 7, and elution with glycine HCl buffer (0.2 mol/L, pH 2.8). Eluted anti-CCR5 Ig was neutralized with 3 mol/L Tris and dialyzed against PBS overnight. F(ab')2 of i.v. Ig were also subjected to affinity chromatography in a similar fashion.
Binding studies of anti-CCR5 Ig
The reactivity of affinity-purified anti-CCR5 Ig with the CCR5 peptide was assessed using an ELISA. Plates were coated with 50 µl of CCR5 peptide (10 µg/ml) in PBS, pH 7.4, for 1 h at 37°C. The wells were washed with PBS-Tween (1) before saturation with PBS-BSA 0.1%. Several dilutions of immunopurified anti-CCR5 Ig were added (100 µl) for 2 h at 37°C. Positive and negative controls consisted of mouse anti-CCR5 mAb 2D7 (10 µg/ml) and of the effluent of the affinity column (anti-CCR5-depleted IgG; 200 µg/ml), respectively. After further washing with PBS-Tween, biotinylated goat anti-mouse IgG, goat anti-human IgG, or goat anti-human F(ab')2 were added for 1 h at 37°C. Following an incubation step with streptavidin, substrate was added and the OD was recorded at 490 nm.
Primary cells and cell lines
The CCR5-GFP-CHO are CHO cells transfected with human CCR5 cDNA fused to a green-fluorescent protein (GFP) vector that encodes a neomycin-resistant gene (15). CCR5-GFP-CHO cells were cultured in Ham’s F12 medium containing FCS (10%) and antibiotics (penicillin, 100 U/ml; streptomycin, 100 µg/ml; and neomycin G418, 800 µg/ml) from Life Technologies (Paisley, U.K.). HeLa-CD4-CCR5-LTRlacZ cells (P4-CCR5) were maintained in DMEM medium containing FCS (10%), penicillin (100 U/ml), streptomycin (100 µg/ml), and puromycin (1 µg/ml). Lymphocytes were obtained by stimulation of PBMC of HIV-1-seronegative healthy individuals with PHA (2.5 µg/ml) for 72 h before stimulation with IL-2 (10 IU) for 24 h. Monocytes/macrophages were obtained by culturing PBMC (107/ml) in RPMI 1640 with heat-inactivated normal human AB serum (10%) in plastic culture plates for 1 h at 37°C. After washing, mononuclear adherent cells were cultured in RPMI 1640 containing FCS (10%), penicillin (100 U/ml), streptomycin (100 µg/ml), and M-CSF (200 U/ml). Monocytes were maintained for 5–7 days to obtain MDM. Purity of the MDM preparation was assessed by morphological criteria and flow cytometry analysis (80–90% CD14 positive and contamination with T cells <3%).
Immunofluorescent staining and cytofluorometry
The CCR5-GFP-CHO and MDM were used to assess the specificity of interaction of purified anti-CCR5 Ig and anti-CCR5 Ig F(ab')2 with membrane-bound CCR5. CCR5-GFP-CHO and MDM were recovered in cold PBS and EDTA (0.02%) for 10 min. Cells (105 in 200 µl PBS/NaN3 0.05%) were incubated with increasing amounts of anti-CCR5 Ig or F(ab')2 (100–500 µg/ml) for 30 min on ice. Anti-CCR5-depleted IgG was used as negative control, and anti-CCR5 mAb 2D7 as positive control. After washing with PBS/BSA (0.2%)/NaN3 (0.05 M), cells were incubated with PE-conjugated goat anti-human Abs for 30 min on ice. Cells were fixed with 1% of paraformaldehyde and analyzed by flow cytometry using a FACSCalibur and the CellQuest software (BD Biosciences).
Chemokine binding and competition assay
CCR5-GFP-CHO cells were collected in Ca2+- and Mg2+-free PBS and 5 mmol/L EDTA, and resuspended in binding buffer PBS/BSA (0.2%)/NaN3 (0.05 M). In a first set of experiments, a saturation curve was established by incubating increasing amounts of human rRANTES (hrRANTES; 1–500 ng/ml) with CCR5-CHO-positive cells for 30 min. The binding of hrRANTES to cells was assessed by FACS analysis using PE-labeled anti-RANTES mAb. Wild-type CHO cells were used to determine nonspecific binding. Competition experiments were then performed using 105 cells in a final volume of 200 µl of PBS/BSA (0.2%)/NaN3 (0.05 M) in the presence of 10 ng/ml hrRANTES and varying amounts of anti-CCR5 Ig or F(ab')2 for 30 min on ice. Unbound RANTES was eliminated by washing with PBS/BSA (0.2%)/NaN3 (0.05 M), and cells were incubated with PE-conjugated anti-RANTES for 30 min on ice. Cells were fixed with 1% paraformaldehyde in PBS. The ability of anti-CCR5 Ig and F(ab')2 to inhibit the binding of RANTES to CCR5-GFP-CHO was analyzed by flow cytometry.
Viral strains
Primary R5-tropic strains YU-2 and JR-CSF, and the X4-tropic strain NDK were a gift from the repository of the Center Integré de Recherches Biocliniques sur le SIDA (Hôpital Saint Joseph, Paris, France).
HIV-1 infection assay
P4-CCR5 (HeLa-CD4+ CXCR4+ CCR5+) cells were infected with the laboratory-adapted HIV-1 strain BaL, the primary R5-tropic HIV-1 strains YU-2 and JR-CSF, and primary X4-tropic HIV-1 strain NDK in the presence of 200 and 500 µg/ml anti-CCR5 Ig. Briefly, cells were cultured in 96 wells (105 cells/ml) in the presence of DEAE-dextran (60 µg/ml) in DMEM/FCS (10%), penicillin (100 U/ml), streptomycin (100 µg/ml), and puromycin (1 µg/ml) for 48 h at 37°C. Cells were infected with appropriate viral dilutions and incubated for 48 h at 37°C. After washing, cells were lysed in 1 M Na2HPO4, 1 M NaH2PO4, 1 M KCL, 1 M MgSO4, 0.5 M EDTA, 50 mM 2-ME, and 10% Nonidet P-40, and CPRG staining was performed by adding the CPRG reagent to cell lysates. The OD was measured at 570 nm. PBL (2 x 106) and MDM (5 x 105) were incubated with anti-CCR5 Ig, anti-CCR5-depleted IgG as negative control (500 µg/ml), or RANTES (500 ng/ml) for 30 min at 37°C before adding virus (BaL, YU-2, and JR-CSF; 20 ng/ml p24). In some experiments, MDM were incubated with anti-CCR5 Ig at 200 and 500 µg/ml, mAb 2D7 (10 µg/ml), or SDF-1 (2.5 µg/ml) before exposure to the BaL HIV-1 strain for 18 h at 37°C. Cells were washed three times, fresh medium was added, and supernatants were collected every 3 days. HIV-1 production was assessed by measuring p24 release using a p24 Ag capture ELISA (HIV-1 core profile ELISA; DuPont de Nemours, Les Ulis, France).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Affinity purification of anti-CCR5 Ig yielded 
0.2% of
total i.v. Ig. We first assessed immunoaffinity-purified anti-CCR5
Abs from i.v. Ig (anti-CCR5 Ig) for reactivity with the CCR5
peptide by ELISA. Mouse mAb 2D7 that interacts with the CCR5 protein
was used as positive control, and the effluent of the CCR5 affinity
column containing anti-CCR5-depleted IgG was used as negative
control. As shown in Fig. 1
, anti-CCR5 Ig bound to the CCR5 peptide in a dose-dependent manner,
whereas Ig depleted of anti-CCR5 Abs failed to recognize the
peptide. Similar results were obtained using
F(ab')2 of natural anti-CCR5 Abs instead of
intact IgG (data not shown). As expected, anti-CCR5 mAb 2D7 bound
to the CCR5 peptide (Fig. 1).
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, purified anti-CCR5 Ig and the
corresponding F(ab')2 bound in a dose-dependent
manner to CCR5-bearing cells. At 500 µg/ml, anti-CCR5 Ig and
F(ab')2 recognized 81% and 57% of CCR5-positive
cells, respectively, whereas the Abs did not bind to untransfected CHO
cells (less than 2%; data not shown). Anti-CCR5-depleted IgG displayed
marginal binding to the CCR5-CHO cells, whereas the anti-CCR5 mAb
2D7 stained 90% of the cells (Fig. 2). MDM express high level of CCR5
molecules (up to 80%). Anti-CCR5 Ig also bound to MDM in a
dose-dependent manner (75% of positive cells at 500 µg/ml) (Fig. 3). A similar level of binding was
observed when using the anti-CCR5 F(ab')2
fraction instead of intact anti-CCR5 Ig (data not shown). In the
latter experiments, mAbs to CCR5 and to CD4 were used as positive and
negative controls, respectively (data not shown).
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hrRANTES bound to CCR5-CHO cells in a dose-dependent manner, and
saturation was reached at 50 ng of hrRANTES, and no binding was
detected when untransfected cells were used. For inhibition
experiments, hrRANTES (10 ng/ml) was incubated with CCR5-CHO cells in
the presence of increasing amounts of anti-CCR5 Ig or the
corresponding anti-CCR5 F(ab')2 (0–1000
µg). Both anti-CCR5 Ig and anti-CCR5
F(ab')2 inhibited the binding of RANTES to
CHO-CCR5 cells in a dose-dependent fashion (Fig. 4). Complete inhibition was observed in
the presence of 500 µg/ml of anti-CCR5 Ig. Abs depleted of
anti-CCR5 IgG exhibited no inhibitory effect (Fig. 4).
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The HeLa P4-CCR5 cells that are CD4+
CXCR4+ CCR5+ were infected
with the BaL strain, the primary R5-tropic HIV-1 strains YU-2 and
JR-CSF, or the primary X4-tropic HIV-1 strain NDK. Infection of the
cells was assessed using 
-galactosidase activity measured by the
CPRG colorimetric test, allowing for the detection of one viral
replication cycle. Inhibition experiments were performed in the
presence of 200 and 500 µg/ml of anti-CCR5 Ig. As shown in Fig. 5, anti-CCR5 Ig inhibited the
replication of BaL (77–81% of inhibition), of the primary R5-tropic
HIV-1 YU-2, and of JR-CSF strains (60 and 64% of inhibition for YU-2
and JR-CSF, respectively). Anti-CCR5 Ig had no effect on infection of
P4-CCR5 with the primary X4-tropic NDK strain. Incubation with RANTES
(500 ng/ml), used as positive control, resulted in a complete
inhibition of viral replication in P4-CCR5 cells infected with BaL,
YU-2, and JR-CSF, whereas RANTES had no effect on infection with HIV-1
NDK. No inhibition of infection was observed in the presence of Ig
depleted of anti-CCR5 Abs (Fig. 5).
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, the production of p24
by infected MDM was significantly inhibited in cultures preincubated
with anti-CCR5 Ig (60% inhibition in case of BaL, 37% for YU-2,
and 64% for JR-CSF) as compared with cultures incubated in the
presence of anti-CCR5-depleted IgG (<5%). In another set of
experiments, RANTES and anti-CCR5 mAb 2D7 inhibited up to 75% the
infection of MDM by BaL, which was similar to the inhibition observed
in the presence of 500 µg/ml anti-CCR5 Ig. Neither SDF-1 nor the
isotypic control for mAb 2D7 had any inhibitory capacity (data not
shown). Anti-CCR5 Ig also inhibited up to 90% the infection of
lymphocytes with BaL, 70% in case of YU-2, and 73% in case of
JR-CSF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NAAbs reactive with self Ags of the IgG, IgM, and IgA isotypes are present in normal serum. NAAbs are encoded by unmutated, germline genes encoding V regions with no or few mutations. NAAbs are characteristically more polyreactive than immune Abs, in the sense that they often recognize several Ags (16). NAAbs exhibit a broad range of affinities, with dissociation constants ranging from 10-5 to 10-8 M (1, 16, 17). Using the surface plasmon resonance technology, we have observed an overall affinity in the micromolar range of natural IgG autoantibodies specific for molecules such as HLA class I, CD4, the RGD motif, and autologous blood group Ags (18, 19, 20). Several functions have been proposed for NAAbs (21), including a role in natural host defense against infection (3).
The presence of anti-CCR5 Abs has previously been documented in
sera of individuals homozygous for a 32-bp deletion in the gene
encoding for CCR5 (CCR5
32), who had been repeatedly exposed to
CCR5-expressing blood cells through sexual activity (22).
Abs from these individuals, in contrast to serum from
CCR5+/+ individuals, competed with radiolabeled
RANTES for binding to the CCR5 receptor and inhibited infection of PBMC
with R5, but not X4, primary isolates of HIV-1 (22). The
role of natural anti-CCR5 Abs in vivo remains unknown. However, a
recent study demonstrated that protection against HIV infection
correlated with the presence of natural anti-CCR5 Abs in the sera
of some HIV-exposed individuals who remain seronegative
(23). The anti-CCR5 Abs down-modulated surface CCR5
expression in vivo and neutralized the infectivity of R5 strains of
HIV-1, providing a basis for the acquisition of resistance to
infection. R5-tropic viral strains are preferentially transmitted via
the mucosal route (24, 25). After crossing epithelial
barrier, virus spreads rapidly, through contact between dendritic cells
and CD4+ lymphocytes cells that favor HIV
replication in the early stage of infection (26, 27, 28).
Hence, blockade of CCR5 with anti-CCR5 Abs could result in the
inhibition of the spreading of R5-tropic viruses in vivo.
CCR5 and CXCR4, the coreceptors of R5-tropic and X4-tropic strains of HIV-1, respectively, represent a potential target to block the entry of virus and infection of lymphocytes and macrophages in vivo. A number of mAbs have been developed in attempt to block the interaction of virus with the CCR5 coreceptor (13, 29). Of note, a murine anti-CCR5 mAb that recognizes the second extracellular loop of CCR5, the major domain for gp120 binding, was shown to block the infection of T lymphocytes with several R5-tropic HIV-1 strains (30). Although murine hybridomas serve as excellent tools for understanding the structure-function relationship between HIV and its coreceptors, they bear obvious limitations from therapeutic point of view. As previously described, i.v. Ig also contain anti-CD4 Abs that block HIV entry (18). The presence of natural anti-CD4 and anti-CCR5 in i.v. Ig that blocks HIV infection allows speculation of the presence of natural Absdirected against the other HIV coreceptors, i.e., CXCR4, CCR3, and CCR2b. Thus, one may consider using i.v. Ig to prepare therapeutically relevant Abs using sequential steps of affinity chromatography.
Because i.v. Ig preparations are in clinical use, our observations suggest that affinity-purified anti-CCR5 Abs from i.v. Ig could be used therapeutically. There is a current interest in the development of anti-CCR5 antagonists for therapeutic use (31). One of the approaches could be the prevention of mother-to-child transmission of HIV. In this context, it is of interest that several combinations of human mAbs against HIV-1 neutralize SHIV-vpu+ (a chimeric simian-human virus) in vitro through synergistic interactions, and that the combination of mAbs protects macaques against i.v. SHIV-vpu+ challenge after delivery (32). In this respect, the identification of natural human Abs with inhibitory capacity that can be isolated from available therapeutic preparations of normal IgG emphasizes the potential of these Abs for passive immunotherapy in HIV infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Srini Kaveri, Institut National de la Santé et de la Recherche Médicale Unité 430, Hôpital Broussais, 96 rue Didot, 75674 Paris Cedex 14, France. E-mail address: srini.kaveri{at}brs.ap-hop-paris.fr
3 Abbreviations used in this paper: NAAbs, natural autoantibodies; CHO, Chinese hamster ovary; CPRG, chlorophenol red--D-galactopyranoside; GFP, green-fluorescent protein; hrRANTES, human rRANTES; MDM, monocyte-derived macrophage; SDF, stromal-derived factor.
Received for publication January 8, 2001. Accepted for publication April 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokines, HIV type 1 second receptors, and exposed uninfected persons. AIDS Res. Hum. Retroviruses 12:1203.[Medline]
32 homozygous individuals: identification of chemokine and HIV-1-blocking human antibodies. Proc. Natl. Acad. Sci. USA 95:5241.This article has been cited by other articles:
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),
i.v. Ig depleted of anti-CCR5 Abs (
; 25–200 µg/ml), and an
anti-CCR5 mAb (2D7) (•; 1.25–10 µg/ml) were incubated with the
peptide immobilized on a microtiter plate. The recognition of CCR5
motif by Ig was assessed by an ELISA, as described in Materials
and Methods.
), F(ab')2 (
). The binding of RANTES to cells was detected using a
PE-conjugated anti-RANTES Ab, and percentage of RANTES-positive
cells was determined by FACSCalibur flow cytometer and the CellQuest
software (Becton Dickinson). A representative of two independent
experiments is depicted in the figure.
), and hrRANTES at 0.5 µg/ml
(