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. 2008 Apr;123(4):508–518. doi: 10.1111/j.1365-2567.2007.02717.x

Inhibition of HIV-1 transmission in trans from dendritic cells to CD4+ T lymphocytes by natural antibodies to the CRD domain of DC-SIGN purified from breast milk and intravenous immunoglobulins

Mary Requena 1, Hicham Bouhlal 1, Nadine Nasreddine 1, Hela Saidi 1, Jean-Chrysostome Gody 3, Sylvie Aubry 4, Gérard Grésenguet 5, Michel D Kazatchkine 6, Rafick-Pierre Sekaly 1,7, Laurent Bélec 1,2, Hakim Hocini 1
PMCID: PMC2433318  PMID: 17999675

Abstract

The present study demonstrates that human breast milk and normal human polyclonal immunoglobulins purified from plasma [intravenous immunoglobulins (IVIg)] contain functional natural immunoglobulin A (IgA) and IgG antibodies directed against the carbohydrate recognition domain (CRD) domain of the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) molecule, which is involved in the binding of human immunodeficiency virus (HIV)-1 to dendritic cells (DCs). Antibodies to DC-SIGN CRD were affinity-purified on a matrix to which a synthetic peptide corresponding to the N-terminal CRD domain (amino-acid 342–amino-acid 371) had been coupled. The affinity-purified antibodies bound to the DC-SIGN peptide and to the native DC-SIGN molecule expressed by HeLa DC-SIGN+ cells and immature monocyte-derived dendritic cells (iMDDCs), in a specific and dose-dependent manner. At an optimal dose of 200 µg/ml, natural antibodies to DC-SIGN CRD peptide purified from breast milk and IVIg stained 25 and 20% of HeLa DC-SIGN+ cells and 32 and 12% of iMDDCs, respectively. Anti-DC-SIGN CRD peptide antibodies inhibited the attachment of virus to HeLa DC-SIGN by up to 78% and the attachment to iMDDCs by only 20%. Both breast milk- and IVIg-derived natural antibodies to the CRD peptide inhibited 60% of the transmission in trans of HIV-1JRCSF, an R5-tropic strain, from iMDDCs to CD4+ T lymphocytes. Taken together, these observations suggest that the attachment of HIV to DCs and transmission in trans to autologous CD4+ T lymphocytes occur through two independent mechanisms. Our data support a role of natural antibodies to DC-SIGN in the modulation of postnatal HIV transmission through breast-feeding and in the natural host defence against HIV-1 in infected individuals.

Keywords: innate immunity, HIV, breast milk, DC-SIGN, dendritic cells, intravenous immunoglobulins

Introduction

Normal human serum contains natural antibodies (Abs) of the immunoglobulin G (IgG), IgM and IgA isotypes that are produced in the absence of deliberate immunization and independently of exposure to foreign antigens (Ags).1 Most natural Abs are self-reactive [natural autoantibodies (NAAbs)]. Recent evidence supports the hypothesis that NAAbs are generated by positively selected autoreactive B cells.2 Several functions have been proposed for NAAbs, including a role in natural host defence against infection and in the control of immune homeostasis.3 The role of NAAbs in immune regulation has been documented in studies of the impact of intravenous immunoglobulin (IVIg) therapy in patients with autoimmune diseases. Treatment of patients for 5 days with very high amounts of IVIg (approximately 400 mg IVIg/kg body weight/day) led to a better clinical prognosis.4,5 IVIg contains large amounts of natural IgG Abs obtained from pools of plasma from thousands of healthy blood donors. Several of the postulated mechanisms of action of IVIg relate to the presence in IVIg of NAAbs to molecules of relevance to the regulation of the immune response. Thus, IVIg has been shown to contain NAAbs directed against several cell surface molecules, including CD4 and CCR5 receptors, which are used by human immunodeficiency virus (HIV) to infect cells.6,7 Indeed, we have recently demonstrated that the breast milk of 80% of HIV-seronegative and HIV-seropositive women contains natural antibodies directed against CCR5 capable of inhibiting infection of macrophages and dendritic cells by R5-tropic HIV.8

Transmission of HIV-1 occurs mainly through mucosal cells, cervicovaginal cells upon sexual transmission, and intestinal cells upon postnatal transmission via breast milk. Transmission of HIV-1 to the infant through breastfeeding is a major cause of new paediatric HIV-1 infections in developing countries. The risk of transmission of HIV-1 through breastfeeding has been reported to range between 5 and 23%, depending on viral load in breast milk and innate and specific immunity.9–11 The mechanisms of protection from infection of infants, who are fed about 700 ml of breast milk per day containing approximately 6 × 105 copies of free virus and 6 × 105 infected cells, are not yet fully understood, although some protecting factors have been identified.12–15 Immature dendritic cells (iDCs) present in the mucosal tissue, together with CD4+ T lymphocytes and macrophages, are among the first immune-competent cells to encounter the virus.16,17 Infectious HIV particles, following capture by iDCs, are transported to the draining lymph nodes where the virus is efficiently transmitted to CD4+ T cells. In the initial phase, HIV interacts with receptors expressed on iDCs such as C-type lectin receptors (CLRs).18 HIV particles bound to the iDC membrane either infect the cell following interaction between the CD4 and HIV coreceptors or are transmitted to CD4+ lymphocytes. The virus may also be internalized and processed for presentation to T cells, or be recycled back to the plasma membrane prior to transmission to CD4+ lymphocytes.19–21 The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) molecule plays a crucial role in binding HIV through the gp120/41 envelope21–24 and in transmitting HIV to target cells. DC-SIGN is a mannose-binding C-type lectin expressed on DCs in mucosal tissue including the rectum, uterus and cervix.25 DC-SIGN is organized as an ectodomain with seven complete and one incomplete copy of a 23-residue motif and a calcium-dependent lectin domain.26 Antibodies to DC-SIGN inhibit the binding of gp120 to DCs and virus transmission.22 The primary ligand site of gp120 on DC-SIGN is located at the top of the C-type lectin domain and includes two Ca2+-binding sites.27 The interaction of gp120 and DC-SIGN is mediated by Ca2+-binding site 2, whereas Ca2+-binding site 1 correctly orients the primary binding site, as evidenced by the blocking effect induced by the DC-SIGN antibodies AZN-D1 and AZN-D2, which recognize Ca2+-binding sites 2 and 1, respectively.27

In the present study, we investigated the presence of natural antibodies in breast milk and IVIg directed to the DC-SIGN CRD domain including gp120 and calcium-binding sites. We demonstrate that breast milk and IVIg contain IgA and IgG antibodies to the DC-SIGN CRD peptide that are capable of binding to the native DC-SIGN molecule expressed on DCs and of inhibiting transmission in trans of R5-tropic HIV-1 to CD4+ T cells. Our results provide evidence for a role of natural antibodies to DC-SIGN CRD in controlling HIV transmission through breast milk and viral spread in the body.

Materials and methods

Antibodies, cells and reagents

IVIg was a gift from Dr S. V. Kaveri (INSERM U681, Paris, France). Breast milk samples from 11 healthy HIV-seronegative mothers were collected at the lactarium of the Institut de Puériculture, Paris (France). Breast milk samples from 13 HIV-1-infected mothers were also collected at the Complexe Pédiatrique in Bangui (Central African Republic). The ethical recommendations of the Ministry of Health of the Central African Republic were followed, including the obtaining of oral informed consent from mothers. Milk samples were centrifuged at 9300 g to separate the cellular, supernatant and lipid fractions. Supernatants were collected and stored at −80° until use. Phycoerythrin (PE)-conjugated anti-CD1a, fluorescein isothiocyanate (FITC)-conjugated anti-DC-SIGN and FITC-conjugated anti-CD14 were obtained from BD Biosciences (San Diego, CA) and PE-conjugated goat anti-human IgA and IgG were obtained from Jackson Immunoresearch (Baltimore, MD). RPMI 1640 (with l-glutamine) was provided by Cambrex (Verviers, Belgium), and penicillin and streptomycin were provided by Invitrogen (Paisley, UK). MSL (medium for separation of lymphocytes) was obtained from PAA (Les Mureaux, France) and fetal calf serum (FCS) was provided by Eurobio (Les Ulis, France). Granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4 and IL-2 were from R & D Systems Europe (Abingdon, UK). PHA was obtained from Sigma Aldrich (St. Louis, MO). The [342–371]-DC-SIGN peptide YWNRGEPNNVGEEDCAEFSGNGWNDDKCNL, which corresponds to the CRD domain, was synthesized by Sigma Aldrich. The CCR5 irrelevant peptide CSSHFPYSQYQFWKNFQTLK, which corresponds to the second extracellular loop of CCR5 (II.E/C-CCR5), was synthesized by the solid phase F-moc method using an Applied Biosystems Model 433A peptide synthesizer (Foster City, CA). The gp120 C-terminus peptide (491–516 LAI) (YKVVKIEPLGVAPTKAKRRVVQREKR) was obtained from the Agence Nationale de Recherches sur le SIDA, France. The gp160 antigen consisted of a purified preparation of baculovirus-expressed recombinant gp160 (rgp160) derived from the envelope of the MN/LAI strain of HIV (kindly provided by Aventis-Pasteur, Paris, France). Sepharose 4B was obtained from Pharmacia Biotech (Geneva, Switzerland). Mannan was purchased from Sigma Aldrich, IgG b12 was a gift from the National Institutes of Health (NIH) and monoclonal anti-DC-SIGN antibody was obtained from R & D Systems Europe (clone 507). The HIV-p24 enzyme-linked immunosorbent assay (ELISA) was obtained from Inngenetics (Gent, Belgium).

HIV strains

The primary R5-tropic strain HIVJRCSF was a gift from Professor F. Barré-Sinoussi (Institut Pasteur, Paris, France). Viral stock produced on IL-2-activated peripheral blood lymphocytes (PBL) was clarified by centrifugation prior to HIV p24 concentration and tissue culture infective dose 50% (TCID50) determination.

Cell line and primary immature monocyte-derived dendritic cells (iMDDCs)

Epithelial HeLa cells positive and negative for DC-SIGN were a gift from Dr O. Schwartz (Institut Pasteur, Paris, France). Cells were maintained in RPMI 1640 supplemented with 1% antibiotics (penicillin/streptomycin) and 10% FCS. Immature dendritic cells were obtained from monocytes differentiated in the presence of GM-CSF and IL-4 for 6 days of culture as previously described.27 Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by Ficoll (MSL) density gradient centrifugation. Monocytes (106 cells/ml) were then cultured in RPMI in plastic tissue culture plates for 45 min at 37°. Following washes, adherent cells were maintained in RPMI, 10% FCS and 1% antibiotics supplemented with IL-4 and GM-CSF (both at 10 ng/ml) to obtain iMDDCs. The medium was changed every 48 hr and new cytokines, IL-4 and GM-CSF, were added to the medium. Contamination of iMDDCs with CD3+ T lymphocytes was < 3% as assessed by fluorescence-activated cell sorting (FACS). iMDDCs were CD1a+, CD86+, CD80+, CCR5+, CD4+, CD83low and DC14– and expressed up to 90% DC-SIGN molecule.

Autologous lymphocytes, corresponding to the non-adherent fraction after the adherence step, were cultured in RPMI, 10% FCS and 1% antibiotics supplemented with phytohaemagglutinin (PHA) (2·5 µg/ml) and IL-2 (10 ng/ml) for 48 hr. Cells were then washed and cultured for an additional 24 hr in the presence of IL-2.

Role of the DC-SIGN CRD peptide in HIV infection

The role of the DC-SIGN CRD peptide in binding gp160 and HIV was investigated by ELISA. Plastic plates were coated overnight at 4° with the DC-SIGN peptide (10 µg/ml) in phosphate-buffered saline (PBS). The plates were washed with PBS/0.1% Tween and saturated with PBS/1% skimmed milk before addition of gp160 (2 µg/ml) in buffer (0·02 m Tris, 0·15 m NaCl and 10 mm CaCl2) supplemented or not with ethylenediaminetetraacetic acid (EDTA). After 4 hr of incubation at 37°, biotin-conjugated IgG to gp160 antibodies (3 µg/ml) was added for 1 hr at 37° before addition of steptavidin for 30 min at room temperature and measure of the optical density (OD) at 490–650 nm. The role of the DC-SIGN CRD peptide in HIV binding was further investigated in an attachment assay. HIVJRCSF particles (10 ng/ml of p24) coincubated or not with DC-SIGN peptide (10 or 50 µg/ml) were added to HeLa DC-SIGN+ cells (250 000 cells/well). After 1 hr of incubation at 37°, cells were extensively washed and lysed, and the HIV p24 level was determined by ELISA.

Purification of anti-DC-SIGN antibodies from breast milk and IVIg

The [342–371]-DC-SIGN peptide was coupled to Sepharose 4B according to the manufacturer's instructions (Pharmacia Biotech). Pools of breast-milk samples from healthy HIV-seronegative women and from IVIg were incubated with the matrix overnight at 4° before extensive washing of the column with PBS until the OD of the effluent reached a value of 0·001. The column was then eluted with 0·2 m glycine-HCl, pH 2·5. The pH of eluted material was rapidly neutralized with 1 m Tris-HCl, pH 8.3, and dialysed against PBS overnight.

Reactivity, specificity and isotype of breast-milk purified anti-DC-SIGN CRD peptide antibodies

Plastic plates were coated with the DC-SIGN peptide at 10 µg/ml in PBS overnight at 4°. The plates were washed with PBS/0.1% Tween prior to saturation with PBS/1% skimmed milk. Dilutions of breast-milk purified anti-DC-SIGN CRD peptide antibodies were then added and incubated for 4 hr at 37°. After washing, peroxidase-labelled goat anti-human F(ab)2 antibodies (2 µg/ml) were added for 1 hr at 37° before addition of peroxydase substrate. As a negative control, the same experiment was carried out using two irrelevant peptides corresponding to 33 amino acids of the CCR5 molecule and to 26 amino acids of the gp120 protein (used at 10 µg/ml).

The isotype (IgG, IgA and IgM) composition of affinity-purified anti-DC-SIGN antibodies was measured by ELISA. Plates were coated with goat anti-human α chain, goat anti-human γ chain or goat anti-human µ chain (all at 3 µg/ml) in PBS overnight at 4°, prior to washing with PBS/0·1% Tween, and saturation with PBS/1% skimmed milk. Serial dilutions of breast milk or IVIg immunopurified anti-DC-SIGN antibodies were then added for 1 hr at 37°. After further washes, goat anti-human F(ab′)2 (2 µg/ml) coupled with peroxidase was added for 1 hr at 37°. After extensive washes, substrate was added and peroxidase activity determined. A pool of normal human sera with known levels of IgG, IgA and IgM was used to obtain standard curves.

An additional experiment was performed to determine the ratio of secretory immunoglobulin (SIg)A1:SIgA2 in total anti-DC-SIGN peptide SIgA purified from breast milk. Plastic plates were coated with the DC-SIGN peptide at 10 µg/ml in PBS overnight at 4°. The plates were washed with PBS/0.1% Tween prior to saturation with PBS/1% skimmed milk. The anti-DC-SIGN SIgA fraction, purified as previously described,28 was then added for 4 hr at 37° before addition of IgA1 protease (0·1 µg/ml) for 2 hr at 37°. After further washes, goat anti-human F(ab′)2 antibody (2 µg/ml) coupled with peroxidase was added for 1 hr at 37° before addition of substrate and quantification of peroxidase activity.

Detection of anti-DC-SIGN antibodies in breast milk of HIV-seropositive and HIV-seronegative women

The presence of anti-DC-SIGN CRD peptide antibodies in breast milk from HIV-seropositive and HIV-seronegative women was assessed by ELISA. Plastic plates were coated overnight at 4° with DC-SIGN CRD peptide (10 µg/ml) in PBS. The plates were washed with PBS/0.1% Tween prior to saturation with PBS/1% skimmed milk. Dilutions of breast-milk supernatants were then added and incubated for 4 hr at 37°. After washing, peroxidase-labelled goat anti-human F(ab′)2 antibodies (2 µg/ml) were added for 1 hr at 37° prior to addition of peroxidase substrate. IVIg purified anti-DC-SIGN CRD peptide antibodies were used as standards. The isotype content of each sample of breast milk was determined by ELISA as previously described and the specific activity was calculated according to the formula:

graphic file with name imm0123-0508-mu1.jpg

Immunostaining and FACS analysis

The expression of cell surface antigens was assessed by cytofluorometry using a FACS Calibur™ and cellquest software™ (Becton Dickinson, San Diego, CA). Cells (1 × 106 cells per test) were incubated with mAbs (5 µg/ml) against membrane molecules for 30 min at 4°, washed with PBS/0·05% NaN3 and fixed with 1% paraformaldehyde prior to analysis of at least 5000 viable events gated by forward and side scatter.

Binding of purified anti-DC-SIGN peptide antibodies to HeLa DC-SIGN-positives cells and to monocyte-derived dendritic cells

HeLa DC-SIGN+ cells, HeLa cells and iMDDCs at day 6 of culture (1 × 104 cells) were incubated with increasing concentrations of purified anti-DC-SIGN peptide antibodies (50, 200 and 400 µg/ml) for 1 hr on ice. Cells were then washed and incubated with FITC-conjugated rabbit anti-human immunoglobulin antibodies for 30 min on ice. The cells were washed, fixed with 1% paraformaldehyde and analysed by FACS. Anti-DC-SIGN mAb (clone 507; R & D Systems Europe) at 10 µg/ml was used as a positive control. As a negative control, anti-DC-SIGN-depleted immunoglobulins (column effluent) were used at the highest concentration (400 µg/ml).

The specificity of the binding was assessed by a competition test using the DC-SIGN peptide. Briefly, natural anti-DC-SIGN antibodies (200 µg/ml) were preincubated at room temperature for 30 min with the DC-SIGN peptide (100 µg) before addition to iMDDCs. After 1 hr on ice, cells were washed and incubated with FITC-conjugated rabbit anti-human immunoglobulin antibodies for 30 min on ice. The cells were then washed, fixed with 1% paraformaldehyde and analysed by FACS.

Inhibition of HIV-1 attachment to HeLa DC-SIGN+ and iMDDCs by natural anti-DC-SIGN peptide antibodies

HeLa DC-SIGN+ cells or iMDDCs (1 × 105 cells) were preincubated with purified anti-DC-SIGN peptide antibodies (50, 200 and 400 µg/ml) for 30 min at room temperature before addition of 10 ng/ml HIVJRCSF p24 for 1 hr at 37°. Cells were then extensively washed and lysed with 0·5% Triton and HIV p24 levels were measured by ELISA. As attachment inhibition controls, anti-DC-SIGN mAb, mannan and column effluent were added to cells prior to the incubation with HIV.

Inhibition of HIV-1 transmission in trans to T cells by anti-DC-SIGN peptide antibodies

iMDDCs (1 × 105 cells) were incubated with 1 ng/ml of HIVJRCSF p24 for 3 hr at 37°. Cells were extensively washed and IL-2-activated lymphocytes (5 × 105 cells) were added at a ratio of 1 : 5 and maintained in coculture in RPMI/10% FCS/1% antibiotics supplemented with IL-2 (10 ng/ml) for 72 hr. Supernatants were then collected and HIV p24 levels in culture medium were measured by ELISA. In inhibition experiments, cells were preincubated with purified anti-DC-SIGN peptide antibodies (at 20, 50 and 200 µg/ml) for 30 min at room temperature before HIV-1 addition. As a positive control, anti-DC-SIGN mAb, mannan or mAb IgG b12 anti-gp160 was added to cells prior to infection with HIV. The same experiment was performed with column effluent used as a negative control.

Statistical analysis

Procedures were based on non-parametric analyses (Mann–Whitney); comparisons between the different groups were made using a two-tailed t-test. Statistical analysis was performed using the spss statistical package (SPSS Inc., Chicago, IL). The level of significance was P = 0·05.

Results

Human breast milk from HIV-1-seronegative and HIV-1-seropositive women contains natural antibodies directed against DC-SIGN

The role of the DC-SIGN CRD peptide in the binding of gp160 and HIV particles to DCs was demonstrated using both an ELISA and an HIV attachment assay. The interaction between gp160 and the DC-SIGN CRD peptide was shown to be specific and calcium dependent (Fig. 1a) and to involve the region of DC-SIGN implicated in HIV-1 attachment to HeLa DC-SIGN+ cells, as demonstrated by the 60% inhibition observed following preincubation of HIV particles with peptide prior to addition to cells (Fig. 1b). We then investigated recognition of the DC-SIGN CRD peptide by immunoglobulins in breast-milk samples from HIV-seronegative and HIV-seropositive women. Eleven of 11 (100%) HIV-negative and eight of 13 (61%) HIV-positive samples contained over 50 µg/ml of anti-DC-SIGN Abs (Figs 2a and b). A higher amount of anti-DC-SIGN CRD peptide antibodies was present in breast milk of uninfected women than in that of infected women (mean 24 versus 9; P = 0·0127) (Fig. 2c).

Figure 1.

Figure 1

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Specificity of the interaction between the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) CRD peptide and human immunodeficiency virus (HIV). (a) An enzyme-linked immunosorbent assay (ELISA) plastic plate, coated with DC-SIGN peptide, was incubated with gp160 in a buffer containing 0·02 m Tris, 0·15 m NaCl and 10 mm CaCl2 supplemented or not with ethylenediaminetetraacetic acid (EDTA). Biotin-conjugated immunoglobulin G (IgG) to gp160 was then added before addition of steptavidin and measurement of the optical density (OD) at 490–650 nm. (b) HIV preincubated or not with DC-SIGN peptide (10 or 50 µg/ml) was added to HeLa DC-SIGN+ cells. After 1 hr of incubation at 37°, cells were washed and lysed, and the p24 concentration was determined by ELISA. Results are expressed as mean ± standard deviation calculated from four independent experiments. Statistical significance as determined by Mann–Whitney test is indicated. *P ≤ 0·05.

Figure 2.

Figure 2

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Anti-dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) CRD peptide reactivity and specific activity in breast-milk samples from HIV-seronegative and HIV-seropositive women. The anti-DC-SIGN CRD peptide reactivities of 11 samples from HIV-negative women (a) and 13 samples from HIV-seropositive women (b) were investigated by enzyme-linked immunosorbent assay (ELISA) as described in the Materials and methods. Results are expressed in μg/ml. (c) Specific activity of the same samples to DC-SIGN peptide was assayed by ELISA as described in the Materials and methods. Specific activity (SA) was calculated as SA = (anti-DC-SIGN antibodies)/(total immunoglobulins) × 100. Results are expressed in arbitrary units (AU). The statistical significance as determined by the Mann–Whitney U-test is indicated.

Antibodies to CRD peptide were immunopurified from breast milk of seronegative women and IVIg and their reactivity against the peptide was then tested by ELISA. Binding of purified antibodies to the peptide was dose dependent (Fig. 3a). Furthermore, the binding of antibodies was specific, as demonstrated by the reactivity against CCR5 and gp120 irrelevant peptides used as controls, which showed less than 15 and 30% cross-reactivity, respectively (Fig. 3b).

Figure 3.

Figure 3

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Dose-dependent and specific binding of antibodies to dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) CRD peptide. (a) Variable amounts (from 25 to 1000 µg/ml) of anti-DC-SIGN CRD peptide antibodies (Abs) purified from intravenous immunoglobulins (IVIg) and breast milk of HIV-seronegative women were incubated with DC-SIGN CRD peptide for 4 hr at 37°. Wells were washed and incubated with goat anti-human F(ab′)2 coupled to peroxidase for 1 hr at 37°. (b) The same experiment was performed with two irrelevant peptides (CCR5 and gp120). Results are expressed as mean ± standard deviation calculated from two independent experiments.

The analysis of breast-milk purified anti-DC-SIGN antibodies showed that 88% of antibodies were SIgA and 6% were IgG (Fig. 4a). However, IgG exhibited a higher specific activity (65%) than SIgA (15%) (Fig. 4b). We also found that, in IVIg, IgG was the predominant isotype (96%) and exhibited the highest specific activity (Figs 4a and c). Human IgA comprises two subclasses of IgA, IgA1 and IgA2, which are present in various proportions in secretions. Breast-milk purified anti-DC-SIGN antibodies were mainly SIgA1 (75%). In contrast, there was no difference in SIgA subclass distribution for anti-CCR5 antibodies purified from breast milk (Fig. 4d).

Figure 4.

Figure 4

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Characterization of purified anti-dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) CRD peptide antibodies. (a) The isotype composition of breast-milk and intravenous immunoglobulins (IVIg) purified anti-DC-SIGN antibodies was evaluated by enzyme-linked immunosorbent assay (ELISA) as described in the Materials and methods. (b, c) The specific activity of breast-milk and IVIg purified anti-DC-SIGN antibodies was assayed as described in the Materials and methods. Specific activity (SA) was calculated as: SA = (anti-DC-SIGN IgA or IgG)/(total IgA or IgG) × 100. Results are expressed in arbitrary units (AU). (d) Determination of secretory immunoglobulin A (SIgA) subclass composition of DC-SIGN and CCR5 antibodies purified from breast milk using an SIgA1 ELISA quantification test. Results are expressed as mean ± standard deviation calculated from two independent experiments. OD, optical density.

Affinity-purified anti-DC-SIGN antibodies from breast milk and IVIg specifically recognize the native DC-SIGN molecule

We investigated the ability of purified anti-DC-SIGN CRD peptide antibodies to bind the native DC-SIGN molecule expressed by HeLa DC-SIGN+ cells and by iMDDCs obtained in the presence of IL-4/GM-CSF for 6 days of culture. iMDDCs were CD1a+, CD86+, CD80+, CCR5+, CD4+, CD83low and DC14– and expressed up to 90% DC-SIGN molecule.

As shown in Fig. 5(a), purified anti-DC-SIGN CRD peptide antibodies bound in a dose-dependant manner to DC-SIGN. Six per cent of HeLa DC-SIGN+ cells were stained with 50 µg/ml of anti-DC-SIGN antibodies purified from breast milk. The level of positive cells increased to a plateau of 25% when purified antibodies were used at 200 µg/ml. Similar data were obtained with purified anti-DC-SIGN antibodies from IVIg. The column effluent used as a negative control at 400 µg/ml stained only 1–2% of cells. Furthermore, only 3% of mock HeLa cells were stained with anti-DC-SIGN purified from both breast milk and IVIg.

Figure 5.

Figure 5

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Cytofluorometric analysis of the binding of purified anti-dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) CRD peptide antibodies to HeLa DC-SIGN+ cells and immature monocyte-derived dendritic cells (iMDDCs). HeLa DC-SIGN+ cells (a) or iMDDCs (b) were incubated with increasing amounts of purified anti DC-SIGN antibodies (50, 200, 400 µg/ml), anti-DC-SIGN monoclonal antibody (mAb) (positive control; 10 µg/ml) and column effluent (400 µg/ml) for 1 hr on ice. Cells were incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-human Ab for 30 min on ice and analysed using FACSCalibur flow cytometer and the cellquest software. (c) Purified anti-DC-SIGN antibodies (200 µg/ml) were preincubated for 30 min at room temperature with DC-SIGN peptide before addition to iMDDCs and binding determination by cytofluorometry. A representative experiment from five independent experiments is shown. MFI, mean fluorescence intensity.

Analysis of binding of breast-milk purified anti-DC-SIGN CRD peptide antibodies to iMDDCs showed that, at 50 µg/ml, 17% of cells stained positively. The percentage of positive iMDDCs increased to a plateau of 32% when purified anti-DC-SIGN antibodies were used at 200 µg/ml, a higher proportion than that obtained with anti-DC-SIGN peptide antibodies purified from IVIg. Indeed, 400 µg/ml of IVIg purified anti-DC-SIGN antibodies were needed to reach a plateau of 30% binding. The column effluent used at 400 µg/ml stained only 1–2% of cells (Fig. 5b).

The specificity of the binding of purified anti-DC-SIGN peptide antibodies to the native DC-SIGN molecule was assessed using a competition test. As shown in Fig. 5(c), when natural antibodies were preincubated with the DC-SIGN peptide prior to addition to cells, the percentage of positive cells decreased from 32 to 22% (31% inhibition) and the mean fluorescence intensity (MFI) decreased from 27 to 12 (56% inhibition).

Natural anti-DC-SIGN CRD peptide antibodies block the attachment of HIV-1 to HeLa DC-SIGN+ cells but not to iMDDCs

We determined the ability of anti-DC-SIGN antibodies purified from breast milk and IVIg to inhibit the attachment of primary R5-tropic HIV-1 to HeLa DC-SIGN+ cells and to iMDDCs. As shown in Fig. 6(a), at 50 µg/ml anti-DC-SIGN antibodies from breast milk inhibited 50% of HIVJRCSF attachment on HeLa DC-SIGN+ cells (132 versus 254 pg/ml; P < 0·001). The inhibition of attachment reached 78% when natural antibodies were used at 400 µg/ml (56 versus 254 pg/ml). Similar data were obtained with anti-DC-SIGN antibodies purified from IVIg. Interestingly, anti-DC-SIGN (clone 507) antibody, used at 400 µg/ml, resulted in 50% inhibition of HIVJRCSF attachment (134 versus 254 pg/ml), indicating that this anti-DC-SIGN mAb had a weaker specific activity than natural anti-DC-SIGN antibodies present in breast milk and in IVIg. Mannan, a major DC-SIGN ligand, used at 200 µg/ml, inhibited attachment by 43%(145 versus 254 pg/ml). HIV attachment to mock HeLa cells represented < 25% of attachment to HeLa DC-SIGN+ cells. The column effluent, used as a negative control, had no significant effect on HIV attachment to HeLa DC-SIGN+ cells.

Figure 6.

Figure 6

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Inhibition of human immunodeficiency virus (HIV) attachment on HeLa dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)+ cells and of HIV transmission in trans from immature monocyte-derived dendritic cells (iMDDCs) to CD4+ T cells by natural anti-DC-SIGN peptide antibodies. (a) HeLa DC-SIGN+ cells were preincubated with different amounts of anti-DC-SIGN antibodies (50, 200, 400 µg/ml) purified from breast milk and intravenous immunoglobulins (IVIg), with mannan (20, 200 µg/ml), with anti-DC-SIGN monoclonal antibody (mAb) (50, 200, 400 µg/ml) or with column effluent anti-DC-SIGN peptide-depleted immunoglobulins (400 µg/ml) before addition of R5-tropic HIVJRCSF for 1 hr at 37°. The cells were then washed and lysed, and p24 antigen was quantified by capture enzyme-linked immunosorbent assay (ELISA). (b) iMDDCs were preincubated with differents amounts of breast milk and IVIg purified anti-DC-SIGN antibodies (20 or 50 µg/ml), mannan (200 µg/ml), anti-DC-SIGN mAb (20 µg/ml) or column effluent anti-DC-SIGN peptide-depleted immunoglobulins (400 µg/ml), before addition of R5-tropic HIVJRCSF for 3 hr at 37°. The cells were washed and autologous CD4+ T cells were added. After 3 days of coculture the production of p24 antigen in culture supernatants was measured. Results are expressed as mean ± standard deviation calculated from five independent experiments for different donors. Statistical significance as determined by the Mann–Whitney U-test is indicated. *P ≤ 0·05.

Similar virus attachment experiments were carried out with iMDDCs. Our results showed that neither natural antibodies to DC-SIGN CRD peptide nor anti-DC-SIGN mAb (clone 507) was able to block significantly HIV-1 attachment to iMDDCs (less than 20%) (data not shown).

Natural anti-DC-SIGN CRD peptide antibodies inhibit transmission in trans of HIV

The effect of natural antibodies on HIV-1JRCSF transmission in trans from iMDDCs to autologous lymphocytes was assessed in a coculture assay (Fig. 6b). Natural antibodies from breast milk inhibited, in a dose-dependent manner, the HIV-1JRCSF transfer from iMDDCs to autologous CD4+ T lymphocytes. At 20 µg/ml, antibodies to DC-SIGN CRD peptide from breast milk and IVIg inhibited virus transfer by 30 and 27% (39 and 41 versus 56 ng/ml HIV p24), respectively. At the plateau of inhibition, natural antibodies inhibited virus transfer by 60% for both breast milk and IVIg purified antibodies (22 and 20 versus 56 ng/ml HIV p24, respectively; P = 0·0016). As a positive control, mAb to DC-SIGN at 20 µg/ml and mannan at 200 µg/ml inhibited transmission in trans of virus to CD4+ T cells by 40% (35 versus 56 ng/ml HIV p24) and 50% (29 versus 56 ng/ml HIV p24), respectively. Neutralizing mAb to gp120 IgG b12 at 20 µg/ml resulted in 75% inhibition (15 versus 56 ng/ml HIV p24; P = 0·0002). As a negative control, no inhibition was observed with the column effluent.

Overall, our results demonstrate that breast milk and IVIg contain natural anti-DC-SIGN antibodies that bind in a specific and dose-dependant manner to the native DC-SIGN molecule expressed by both HeLa DC-SIGN+ cells and iMDDCs. Purifed anti-DC-SIGN antibodies were able to inhibit both the attachement of the HIV particle to the HeLa DC-SIGN+ cells and the transfer of the HIV from iMDDCs to autologous T lymphocytes.

Discussion

In the present study, we have demonstrated the presence of natural antibodies directed against the DC-SIGN CRD domain in human breast milk and in IVIg and their ability to inhibit HIV transmission in trans from dendritic cells to autologous CD4+ T cells. The antibodies were purified by affinity chromatography on the DC-SIGN CRD synthetic peptide which includes Ca2+-binding site 2, which is involved in HIV gp120 binding, and Ca2+-binding site 1, which stabilizes the binding site. Both Ca2+-binding sites are important for DC-SIGN function as chelating agents block the interaction of DC-SIGN with its ligands.27 The DC-SIGN CRD peptide showed a similar calcium-dependent activity in that its interaction with recombinant gp160 was blocked by EDTA. Preincubation of the virus with CRD peptide was shown to inhibit the binding of HIV to HeLa DC-SIGN+ cells in a dose-dependent manner.

Natural antibodies to the DC-SIGN CRD peptide were affinity-purified from breast milk of HIV-seronegative women and IVIg. Their weak reactivity against two irrelevant peptides (gp120 and CCR5 peptide) confirmed their polyreactivity. The natural antibody reponse to DC-SIGN in breast milk differed from that in IVIg. Thus, natural antibodies to DC-SIGN CRD were predominantly of the IgG class in IVIg, whereas they were predominantly of the SIgA isotype in breast milk. Furthermore, the specific activities of natural antibodies to DC-SIGN CRD were higher for IgG than for SIgA in breast milk. These results are consistent with the concept of compartmentalization of the humoral immune response in breast milk, as previously demonstrated for gp160 and CCR5 antigens.7,8,29

Breast-milk anti-DC-SIGN antibodies were mainly of the SIgA isotype. The mean concentration of natural anti-DC-SIGN antibodies in breast milk was 3 times higher than that of natural antibodies to CCR5. In addition, 75% of natural breast-milk SIgA to DC-SIGN CRD peptide and only 50% of that to CCR5 were shown to belong to the SIgA1 subclass. The biological significance of SIgA1 and SIgA2 subclasses remains unclear, but is thought to be associated with the nature of recognized antigens.30 One may hypothezise that the SIgA1 subclass predominance of natural antibodies to DC-SIGN CRD peptide may relate to the presence of one site of glycosylation in the area of the DC-SIGN lectin corresponding to the DC-SIGN CRD peptide,27 and to the polytropic functionalities of the DC-SIGN lectin.21 Furthermore, SIgA1 is susceptible to cleavage by bacterial proteases on mucosal surfaces, which are frequently colonized with IgA1 protease-producing bacteria. Hence, it is likely that IgA1 and IgA2 antibodies differ in their protective roles because of their different susceptibility to bacterial IgA1 proteases. However, in our conditions, both whole SIgA directed to DC-SIGN peptide and its F(ab′)2 fragments were capable of inhibiting infection in trans of autologous T cells. Taken together, these observations demonstrate the diversity of the natural antibody response in breast milk, in terms of antibody production, isotype specificity and, probably, antigen-dependent functionality.

The content of natural antibodies to the DC-SIGN CRD peptide in breast milk was compared between HIV-infected and HIV-seronegative mothers. One hundred per cent of samples from HIV-seronegative women and 61% of samples from HIV-seropositive women exhibited more than 50 µg/ml of anti-DC-SIGN CRD peptide antibodies, corresponding to the concentration needed to inhibit 50% of HIV transmission in trans from dendritic cells to autologous CD4+ T lymphocytes. Natural antibodies to DC-SIGN CRD peptide purified from breast milk of HIV-seronegative women exhibited higher specific activity than antibodies purified from breast milk of HIV-infected women. The possibility exists that HIV may escape the inhibiting effect of the anti-DC-SIGN natural antibodies in vivo by down-modulation of the level and avidity of natural antibodies to DC-SIGN. Indeed, the expression of dendritic cell markers is reduced during HIV infection, for example for dendritic cell-lysosomal associated membrane protein (DC-LAMP) mRNA in lymph nodes and spleen and DC-SIGN in spleen, as observed during simian immunodeficiency virus (SIV) infection in macaques.31 The down-regulation of DC-SIGN expression in HIV infection could limit the expansion of B lymphocyte clones producing natural antibodies directed to DC-SIGN, while the remaining B clones could only produce low-avidity antibodies.

Breast-milk and IVIg purified antibodies to DC-SIGN CRD peptide were shown to bind specifically to the native DC-SIGN expressed by HeLa DC-SIGN+ cells and by iMDDCs. The binding of anti-DC-SIGN antibodies was specific and dose dependent and decreased when the natural antibodies were preincubated with the DC-SIGN peptide prior to incubation with cells. The apparently weak binding to native DC-SIGN expressed by dendritic cells of natural antibodies to the DC-SIGN CRD peptide, by comparison with that observed for monoclonal antibodies to DC-SIGN, indicated the low affinity of natural antibodies for their antigen. Hence, the partial inhibition of the binding observed after preincubation with the DC-SIGN peptide could also be explained by the fact that natural antibodies are induced in vivo against the peptide in the conformation of the native DC-SIGN molecule.

Transmission of HIV-1 occurs mainly through mucosal epithelial cells, cervicovaginal cells upon sexual transmission, and intestinal cells upon postnatal transmission via breast milk. HIV is transmitted as either as free or cell-associated virus. The majority of CD4+ cells in colostrum are CD14+ macrophages expressing both chemokine receptors and DC-SIGN when stimulated with IL-4.32 The presence of DC-SIGN on mucosal cells and its ability to efficiently bind and transmit the virus are considered to be crucial for HIV mucosal transmission. We thus evaluated the ability of natural antibodies to DC-SIGN CRD peptide purified from breast milk and IVIg to block the transmission in trans of HIV from dendritic cells to CD4+ T lymphocytes. Affinity-purified anti-DC-SIGN CRD peptide antibodies were able to inhibit HIV transmission in trans in a similar manner to that observed with the highly neutralizing IgG b12 monoclonal antibody directed to gp120 and with mannan molecules (60% inhibition versus 75 and 50%, respectively). These results demonstrate that DC-SIGN CRD peptide recognized by natural antibodies is involved in HIV transfer from iDCs to T cells. We further observed that natural antibodies to DC-SIGN peptide inhibited in a dose-dependent manner the attachment of HIV-1 to HeLa DC-SIGN+ cells, as previously reported.22,33 In contrast, neither anti-DC-SIGN antibodies purified from breast milk and IVIg nor monoclonal anti-DC-SIGN antibodies inhibited HIV-1 attachment to dendritic cells, as recently reported by Granelli-Piperno and colleagues.34 These results contrast with the 80% inhibition of HIV attachment to iDCs by Abs directed to DC-SIGN or by DC-SIGN RNA interference treatment reported by others.22,35,36 Discrepancies between studies are probably attributable to the use of soluble recombinant gp120, rather than whole HIV particles, to carry out dendritic cell attachment assays.

Interestingly, protection against HIV-1 infection was shown to correlate with the presence of natural anti-CCR5 Abs in sera of some HIV-exposed but uninfected individuals.37 These CCR5-reactive antibodies in seronegative partners of HIV-seropositive individuals down-modulate surface CCR5 and neutralize the infectivity of R5 strains of HIV-1 in vitro. We now demonstrate that breast milk and IVIg contain natural antibodies capable of binding to the native DC-SIGN CRD peptide and of inhibiting transmission of HIV-1 to autologous CD4+ T lymphocytes, providing further evidence that natural antibodies may be involved in the negative modulation of both postnatal HIV transmission through breast-feeding and virus spread in infected individuals.

Acknowledgments

The study was supported by the University Paris V, Institut Nationale des Sciences et de la Recherche Médicale (INSERM), and Agence Nationale de la Recherche sur le SIDA et les Hépatites Virales. HS was the recipient of a PhD fellowship from the VIth PCRD (Workpackage 15 of the EMPRO programme); NN was the recipient of a PhD fellowship from the Fondation pour la Recherche Médicale, Paris; MR was the recipient of a PhD fellowship from SIDACTION; HB and HH were supported by the Agence Nationale de la Recherche sur le SIDA et les Hépatites Virales (ANRS), Paris, France. We would like to thank the staff of the Complexe Pédiatrique in Bangui, Central African Republic, for their participation in the study, and also the HIV-infected mothers who volunteered to be included in this research. We also thank Dr S. Aubry from the Institut de Puériculture, Paris, for providing milk samples from healthy mothers, Professor F. Barré-Sinoussi of the Institut Pasteur, Paris, for providing HIV-1 strains, and Drs Olivier Schwartz and Srinivas Kaveri for the gift of HeLa/HeLa DC-SIGN cells line and IVIg, respectively. We finally thank Dr Vladimira Donkova-Pitrini, Laboratoire d'Immunologie Biologique, Hôpital Européen Georges Pompidou, Paris, and Ms Sandrine Moussa, Institut Pasteur of Bangui, Central African Republic, for their excellent technical assistance.

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