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The Journal of Immunology, 2001, 166: 6257-6265.
Copyright © 2001 by The American Association of Immunologists

Secretory IgA Specific for a Conserved Epitope on gp41 Envelope Glycoprotein Inhibits Epithelial Transcytosis of HIV-11

Annette Alfsen, Pierre Iniguez, Edwige Bouguyon and Morgane Bomsel2

Institut National de la Santé et de la Recherche Médicale, Unité 332, Institut Cochin de Genetique Moleculaire, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As one of the initial mucosal transmission pathways of HIV (HIV-1), epithelial cells translocate HIV-1 from apical to basolateral surface by nondegradative transcytosis. Transcytosis is initiated when HIV-1 envelope glycoproteins bind to the epithelial cell membrane. Here we show that the transmembrane gp41 subunit of the viral envelope binds to the epithelial glycosphingolipid galactosyl ceramide (Gal Cer), an alternative receptor for HIV-1, at a site involving the conserved ELDKWA epitope. Disrupting the raft organization of the Gal Cer-containing microdomains at the apical surface inhibited HIV-1 transcytosis. Immunological studies confirmed the critical role of the conserved ELDKWA hexapeptide in HIV-1 transcytosis. Mucosal IgA, but not IgG, from seropositive subjects targeted the conserved peptide, neutralized gp41 binding to Gal Cer, and blocked HIV-1 transcytosis. These results underscore the important role of secretory IgA in designing strategies for mucosal protection against HIV-1 infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mucosal surfaces are the major site for HIV-1 entry (1). HIV-1 transmission occurs through exposure of mucosal surfaces to HIV-1-infected fluids, such as semen, colostrum, breast milk, and cervicovaginal fluid (2, 3). Mucosal Abs in seropositive subjects consistently contain S-IgA specific for HIV-1 envelope glycoproteins (4). Studies in both macaques and humans (5, 6) suggest that HIV-1 mucosal Abs participate in host resistance to infection. Indeed, HIV-1-specific S-IgA has been detected in mucosal fluids of uninfected individuals having unprotected sexual intercourse with HIV-1-seropositive partners, including sex workers and sero-different couples (7, 8, 9).

We previously showed that HIV-1 can cross the epithelium in vitro by transcytosis across epithelial cells, the most abundant cell type at mucosal surfaces (10, 11). In these studies, we reconstituted the first step of HIV-1 infection in an in vitro model using a monostratified epithelial cell line barrier. PBMCs infected with HIV-1 primary isolates (HIV-1+ PBMCs) adhered to the apical surface of the epithelial cells (12), establishing a microenvironment similar to the "immunological synapse" at the dendritic cell-CD4+ T lymphocyte interface during Ag presentation (13). Contact between HIV-1-infected PBMCs and epithelial cells induces massive polarized budding of HIV-1 in the microenvironment of the synapse. The newly formed virus is rapidly internalized by the epithelial cells, transcytosed to the basolateral pole of the cell, and released as infectious virions into the basolateral (serosal) environment where the virions could infect lamina propria mononuclear cells. The relevance of this in vitro model using human transformed cell lines as target cells to in vivo early mechanism of HIV-1 entry was confirmed using specimens of human mucosal tissue (11), as infection in human is absolutely nonethical.

Two molecules are required for the transcytosis of HIV-1 across CD4-negative epithelial cells (10): the HIV-1 surface envelope glycoprotein subunit gp120 and glycosphingolipid galactosyl ceramide (Gal Cer),3 which is thought to act as gp120 epithelial cell receptor (14, 15). Gal Cer is markedly enriched at the apical surface of epithelial cells (16) and participates in the establishment of microdomains referred to as rafts, which act as platforms for endocytosis (17, 18) and transcytosis (19, 20).

Here we show that a conserved epitope on gp41, the transmembrane subunit of the HIV envelope glycoprotein, mediates gp41 binding to epithelial cell Gal Cer and that S-IgA neutralizes this binding, thereby preventing HIV-1 transcytosis across epithelial cell monolayers. In addition, we identify a conserved epitope on gp41 to which the neutralizing S-IgA is directed and that this conserved epitope is immunodominant for the humoral mucosal response in HIV-seropositive patients. Finally, we demonstrate that both the binding of HIV-1 envelope glycoproteins to Gal Cer and the organization of Gal Cer in raft microdomains is required for effective HIV-1 transcytosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

Cervicovaginal secretions were collected from eight HIV-1(clade E)-seropositive women at the Institut Pasteur de Pnom Phen, Cambodia. Secretions were collected after 2 days of sexual abstinence using 3 ml of sterile PBS. Samples were centrifuged and frozen and stored at -80°C. Sample contamination by sperm was measured using the seminal fluid detection assay (SEMA; Humangen Fertility Diagnostics, Charlottesville, VA) according to the manufacturer, but optimized by increasing the incubation times and using o-phenylenediamine dihydrochloride as substrate. Contaminated samples were discarded. Colostrum samples were collected from five HIV-1(clade A)-infected women aged 25–40 years from the Institut Pasteur de Bangui, Cambodia (21). All subjects were at non-AIDS stages of HIV infection. Control specimens consisted of two pools of 10 colostrums from healthy seronegative volunteers. S-IgA and IgG were purified as we described earlier (21). 2F5 is a human monoclonal IgG (26). Monoclonal human IgGs C31 anti-gp120 and D1 anti-gp41 used in ligand blotting experiments were obtained from immortalized PBMCs of an HIV-1-seropositive patient (62, 63).

Antigens

Recombinant HIV-1 envelope glycoprotein gp160 HIV-1 MN/LAI (VV.TG.9150) was obtained from Pasteur-Mérieux Sérum et Vaccins (Lyon, France) or from Advanced BioScience Laboratories (Kensington, MD). Recombinant HIV-1-gp 41 and gp120 were obtained from Intracell (Cambridge, MA).

ELISA

ELISAs were performed as described previously (21). Briefly, plates were coated overnight at 4°C with 100 µl/well of either recombinant HIV-1 envelope glycoprotein gp160 (2.5 µg/ml) or HIV-1 peptides (0.2 µg/ml) diluted in PBS. After blocking the plates with 3% skimmed milk and washing, purified S-IgA or IgG were added and incubated for 1 h at 37°C. After washing, rabbit HRP-labeled Abs to human F(ab')2 (1/2000) were added for 1 h at 37°C. HRP activity was detected with o-phenylenediamine (Sigma, St. Louis, MO) and read at 492 nm with a Titertek Multiskan spectrophotometer (Flow Laboratories, Glasgow, U.K.). The results were compared with a standard curve of a pool of whey from human milk containing 460 µg/ml of S-IgA. A large pool of 250 normal human sera containing 14 mg/ml of IgG was used as serum standard. The specific activities of S-IgA and of IgG against gp160 were expressed as arbitrary units per mg of Igs.

The ratio of S-IgA/IgG anti-gp160 = S-IgA specific activity for gp160/IgG specific activity for gp160 represents the relative amount of Abs in the two Ig subclasses. For the peptide specific activity analysis, the results for each colostrum sample and cervicovaginal secretion (#) are given by the ratio of S-IgA/IgG anti-peptide = Qi/ratio of S-IgA/IgG anti-gp160, where Qi = S-IgA specific activity for peptide i/IgG specific activity for peptide i is the ratio of the specific activity of S-IgA to that of IgG for the various HIV-1 envelope glycoprotein peptide i. Therefore, the ratio of S-IgA/IgG anti-peptide is Qi normalized to the total amount of anti-HIV-1 envelope glycoprotein Abs contained in S-IgA subclass relative to IgG.

HIV-1+ PBMC

HIV-1+ PBMC were prepared as described previously (11). Primary isolates were obtained by culture of PBMCs from HIV-1-infected Cameroonese women (subtype A, nonsyncytia-inducing phenotype) and from HIV-1-infected Italian mothers (subtype B, syncytia-inducing and nonsyncytia-inducing phenotypes) (64).

Epithelial cell transcytosis assay and neutralization of transcytosis by S-IgA and IgG or by methyl-{beta}-cyclodextrin (m{beta}CD)

HIV-1 transcytosis across epithelial cells and neutralization of transcytosis by Abs was performed as previously described (21). Briefly, the endometrial cell line HEC-1 or intestinal cell line HT-29 clone 19 (65) cells expressing apical Gal Cer (data not shown) were each grown as a tight, polarized monolayer for 7 days on a permeable filter support (0.45-µm pore size) forming the interface between two independent chambers, the upper one bathing the apical (luminal) surface of the epithelial monolayer, and the lower one bathing the basolateral (serosal) surface. Purified colostrum S-IgA and IgG (20 µg), or the 2F5 IgG (.1 µg) were added to the apical chamber and incubated for 10 min at 37°C. To initiate virus transcytosis, 2 x 106 HIV-1+ PBMCs were added to the apical chamber. Contact between HIV-1+ PBMCs and the epithelial cell monolayer resulted in rapid budding of the HIV-1-virions, followed by their transcytosis from the apical to the basolateral pole of the epithelial cells. After 2 h, inhibition of transcytosis by Ab was determined by detection of p24 in the basolateral medium by ELISA (Pasteur Sanofi, France). After 2 h, the level of p24 in the absence of Ab or in the presence of control nonimmune clostral S-IgA or IgG was measured and found to be similar with a value of 150 pg/ml. This value was taken as 100% of transcytosis and used to express the results. When indicated, Ig was preincubated with the ELDKWA peptide to block the recognition site on the Ig molecule as described (11). As control, an irrelevant ELDKWA scrambled peptide was used. After overnight binding at 4°C, remaining unbound peptide was dialyzed out before addition of the Ig fraction as described above. Assays were performed in duplicate for each of the variables: HIV subtype (PBMCs infected with HIV-1 subtype A or B subtypes) and cell lines (HT29 or HEC-1), which is between four and ten different conditions. Interassay variation was <10%. Fig. 2Go shows the mean values of these combined experiments.



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  		FIGURE 2. ELDKWA gp41 peptide is a major epitope involved in inhibition of HIV-1 transcytosis by colostrum S-IgA but not by colostrum IgG. Purified clostral S-IgA (20 µg) and IgG (20 µg) were preincubated overnight at 4°C with (+) or without (-) the ELDKWA gp41 peptide. After dialysis to remove unbound peptide, the Ig samples were added to the mucosal (apical) medium overlying the tight monolayer of epithelial cells, before addition of HIV-1+ PBMCs as the source of primary HIV-1 viruses. After a 2-h incubation at 37°C, the extent of viral transcytosis was evaluated from the serosal (basolateral) medium by detection of HIV-1 p24 Ag. The results are expressed as the percentage of transcytosis compared with the standard in which control clostral S-IgA and IgG were added to the apical medium. As reference, serum-derived IgG 2F5 Ab (.1 µg) specific for ELDKWA and known to inhibit infection of CD4+ primary cells by a large variety of HIV-1 primary isolates was similarly analyzed. Data are expressed as mean ± SEM from at least nine independent experiments. n.s., Nonspecific.

 
To analyze the role on HIV-1 transcytosis of the cholesterol-depleting drug m{beta}CD (Sigma), optimal drug concentration and incubation time were determined using previously described conditions (29, 66, 67). Tight HT29 cell monolayers were preincubated first for 30 min at 37°C in RPMI 1640 supplemented with 0.25% fatty acid-free BSA (Boehringer Manheim, Mannheim, Germany). m{beta}CD was then added at 1, 5, and 10 mM, and the cell monolayers were incubated up to 4 h at 37°C. Transepithelial resistance (TER) was measured every hour (10). Whereas no significant differences between the control (no m{beta}CD) and drug-treated monolayer could be detected after 1-h treatment with 10 mM m{beta}CD (110 ± 5 ohm x cm2), TER started gradually to decrease, reaching 10 ± 4 ohm x cm2 after 4-h incubation (data not shown). m{beta}CD (5 mM) gave only a 10–15% reduction in TER. Full reversal of these effects were observed for all conditions after washing out the drug and reculturing for 12 h in RPMI 1640 supplemented with 10% FCS, except for incubation for 4 h with 10 mM m{beta}CD when TER did not recover. For these studies, epithelial monolayers were pretreated for 1 h at 1–10 mM m{beta}CD, cells then were washed free of drug, and transcytosis was assayed. TER was measured at the end of the assay and was not significantly different from the values under standard conditions (110 ± 5 ohm x cm2). The results are expressed as the mean percentage of the transcytosis inhibition obtained by at least three independent experiments performed in duplicate as compared with standard conditions in the absence of drug. To test the reversibility of the cholesterol depletion, after 1 h of drug treatment, the cells were washed free of drug and recultured for 12 h before transcytosis was measured.

Binding of recombinant envelope glycoprotein to epithelial cells

Recombinant envelope glycoproteins, gp41 or gp120 (4 nM) were added to the apical pole of confluent, tight monolayers of HT29 or HEC-1 epithelial cells pre-equilibrated in RPMI 1640, 10% FCS at 12°C. The interaction of proteins with epithelial cells was performed first at 12°C for 30 min to maximize the binding without incurring endocytosis. Then, the epithelial monolayer system was transferred to 37°C for 8 min to allow proper organization of raft microdomains and firm attachment of the recombinant viral envelope glycoproteins to the cells. Next, the cell system was transferred to 4°C, and unbound proteins were removed by extensive washing (4x) for 30 min. Cell monolayers were then fixed with 4% paraformaldehyde, processed for immunofluorescence, and analyzed by confocal microscopy as described previously (11). Primary Abs were the specific mAbs D1, directed against gp41 and C31, directed against gp120 (both at 5 µg/ml). Images presented are projections of consecutive sections (<10, with a step of 0.5 µm between two consecutive sections) corresponding to the apical domain of the epithelial cells above tight junctions (21). When indicated, a 100 molar excess ELDKWA or anti-Gal Cer mAb (68) at a concentration known to inhibit HIV-1 transcytosis (21) was added to the apical medium 10 min before addition of recombinant proteins. Addition of an irrelevant peptide at similar concentration into the apical medium did not affect the binding pattern of recombinant HIV-1 envelope glycoproteins (data not shown).

Ligand blotting

Ligand blotting was performed as previously described (69). Briefly, lipids, Gal Cer (Avanti Polar Lipids, Alabaster, AL), monosialoganglioside GM1 (Sigma), or phosphatidylcholine (PC; Serdary Research Laboratories, London, Ontario, Canada) were dissolved in chloroform/methanol (1:1) at 5 mg/ml and spotted onto nitrocellulose (NC) supports (Bio-Rad, Hercules, CA). The NC strips were incubated for 1 h at room temperature in PBS containing 8% BSA to saturate nonspecific binding sites. Recombinant gp160, gp41, or gp120 dissolved at 4 nM in PBS containing 1% BSA were allowed to bind to immobilized lipids for 4 h at room temperature. After extensive washing in PBS containing 1% BSA and 0.05% Tween 20, ligand binding was detected with specific mAbs to gp41 (D1) or SU-gp120 (C31) (5 µg/ml) for 16 h at 4°C followed by the Vector Super ELITE kit (Burlingame, CA) as described by the manufacturer using goat anti-mouse biotinylated mAb and avidin-HRP. Finally, HRP activity was detected with ECL (Amersham, Little Chalfont, U.K.) and autoradiography on Amersham RNP6H film. Exposure time of the autoradiograms was <1 min. For CT-B (the B subunit of cholera toxin) ligand blotting (27), biotinylated CT-B (Sigma) at 2 µg/ml was used, and detection was conducted with streptavidin as described above. When indicated, clostral IgG or S-IgA (#63 or 64) were added together with the viral glycoproteins. In some cases, the various Ig were preincubated with the ELDKWA peptide binding in an attempt to block the corresponding recognition site on the Ig molecule. As results obtained in the presence of the two Ab samples were identical, only experiments with colostrum #63 are shown on Fig. 4Go.



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  		FIGURE 4. gp41 binds to Gal Cer in an interaction involving the ELDKWA epitope that is blocked by specific S-IgA. a, For ligand blotting experiments, various lipids (Gal Cer, GM1, and PC) were spotted on NC. One of the following ligands, Rec gp160, transmembrane gp41 subunit (TM-gp41), surface gp120 subunit (SU-gp120), or CT-B was allowed to bind to immobilized lipids in the presence (+) or absence (-) of an excess of the ELDKWA peptide. Rec gp160, gp41, and gp120 bind specifically to Gal Cer, but only the binding of gp41 was blocked by the ELDKWA peptide (arrow). b, Binding of gp41(TM-gp41) and gp120 (SU-gp120) to immobilized Gal Cer was performed in the presence of S-IgA of patient 63 or the paired IgG, which had been preincubated (+) or not (-) with an excess of the ELDKWA peptide. Only the binding of gp41 was displaced by S-IgA (arrow), and this inhibition was blocked by preincubating the S-IgA with the ELDKWA peptide (lane below arrow).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clostral and cervicovaginal S-IgA to the ELDKWA motif of gp41

We previously reported that clostral IgG and S-IgA from HIV-1-seropositive women bind to the HIV-1 envelope glycoprotein precursor gp160 (21). We now investigate the epitope(s) on gp160 that were recognized by the S-IgA and Ig, using Abs from both colostrum and cervicovaginal secretions (Fig. 1Go, b and d). The specific activity of S-IgA and IgG for four epitopes on the HIV-1 envelope glycoproteins implicated in HIV-1 infection was analyzed. Two epitopes were on gp120: a V3 loop (MN isolate) hypervariable region and the C5 constant region (22) shown to be associated with neutralization or with slow HIV-1 progression. The other two epitopes corresponded to conserved gp41 motifs: the principal immunodominant domain (PID) (23) and the ELDKWA motif. The ELDKWA motif (aa 662–667) is a conserved epitope recognized by human serum IgG 2F5, the only Ig thus far identified that is directed against the gp41 and capable of neutralizing infection by a large majority of primary HIV-1 isolates (24). ELDKWA is part of the larger peptide DP178 (also called T-20) that has been shown to inhibit HIV-1 infection at nanomolar concentrations in cell culture experiments and to have an antiviral effect in a phase I clinical trial in humans (25). We previously showed that this epitope was used by both dimeric IgA and IgM isolated from HIV-1-seropositive patients to neutralize intracellularly HIV-1 transcytosis by epithelial cells (11). The results in Fig. 1Go clearly indicate that for each of the five colostrums and seven of eight cervicovaginal secretions tested, S-IgA anti-HIV-1 envelope bound mainly to the ELDKWA epitope of gp41. In contrast, IgG anti-HIV-1 envelope from the same specimen of secretion failed to bind this gp41 domain. For the one cervicovaginal sample (Fig. 1God, I18), the amount of S-IgA specific for ELDKWA was still significant and 13 times more abundant than the paired IgG. This difference in Ig subclass binding to ELDKWA peptide is due to a higher S-IgA and lower IgG reactivity for each sample. In contrast, the reactivity for gp160 was always higher for IgG than for IgA (Fig. 1Go, a and c). Indeed, the ratio of clostral S-IgA anti-gp160 to clostral IgG anti-gp160 was 0.015–0.1 (Fig. 1Goa), indicating a much lower quantity of S-IgA than IgG specific for HIV envelope glycoprotein in colostrum. A similar analysis of cervicovaginal S-IgA and IgG confirmed the ratio (ratio of S-IgA/IgG anti-gp160 <1) (Fig. 1Goc), indicative of the higher IgG anti-gp160 content as compared with S-IgA anti-gp160 in mucosal secretions.



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  		FIGURE 1. The ELDKWA peptide from gp41 is a major epitope recognized by clostral and cervicovaginal S-IgA of HIV-1-seropositive women. For each clostral (a) and cervicovaginal (c) sample (#), S-IgA and IgG binding activity to HIV-1 envelope glycoprotein gp160 was compared. The relative amount of anti-HIV-1 envelope glycoprotein Abs contained in each Ig subclass sample is given by the ratio of S-IgA/IgG anti-gp160 calculated as in Materials and Methods. For each clostral (b) and cervicovaginal (d) sample (#), specific activities of S-IgA and IgG binding to various HIV-1 envelope glycoprotein peptides (i) were compared and are given by the ratio of S-IgA/IgG anti-peptide calculated as described in Materials and Methods.

 
Transcytosis of HIV-1 across a tight epithelial barrier is impaired by S-IgA against the ELDKWA motif of gp41

We next investigated whether polyclonal S-IgA and IgG purified from the colostrum of HIV-1-seropositive women could neutralize transcytosis of HIV-1. For these experiments, we used our previously described transcytosis assay and PBMCs acutely infected with primary isolates of HIV-1 of subtype A or B (HIV-1+ PBMCs). The ability of mucosal Ig to inhibit transcytosis of HIV-1 was higher for S-IgA (80% mean reduction) than for IgG (49% mean reduction) (Fig. 2Go). The amount of virus produced at the apical pole of the epithelial barrier during the assay was similar in the absence or presence of the Abs (data not shown), indicating that the Abs did not alter PBMC virus production. In addition, no difference in neutralization of transcytosis was detected using PBMCs acutely infected with HIV-1 isolates of subtype A or B. Clostral S-IgA and IgG were purified from seropositive women infected with HIV-1 subtype A but could equally neutralize transcytosis of primary isolates of subtypes A, B, and D. This strongly suggested that the epitopes recognized by the neutralizing Abs are conserved regions of HIV-1 envelope glycoprotein. No difference in transcytosis could be detected using HEC-1 endometrial or HT29 (clone 19) intestinal cell lines. Such inhibitory efficiency for S-IgA even though not complete, is biologically significant, considering that the Ig samples were polyclonal; consequently, titration of specific Ab-mediated inhibition could not be performed.

Because S-IgA but not IgG from the different colostrums (Fig. 1Gob) and cervicovaginal secretions (Fig. 1God) strongly recognized the ELDKWA peptide, we investigate whether this epitope was recognized by the colostrum Igs that neutralized HIV-1 transcytosis.

In experiments designed to block neutralization, equal amounts of purified IgG and S-IgA were preincubated with ELDKWA peptide or with a ELDKWA scrambled irrelevant peptide for control (Fig. 2Go). After dialysis to eliminate excess free peptide, the Ig samples were assayed for their capacity to neutralize HIV-1 transcytosis. Neutralization was significantly decreased when S-IgA, but not IgG, had been preincubated with the ELDKWA peptide implicating ELDKWA as key peptide involved in S-IgA inhibition of HIV-1 transcytosis. Transcytosis neutralization was not inhibited by an excess of peptide inadequately removed by dialysis because under the conditions referred to as "standard + ELDKWA" (Fig. 2Go), HIV-1 transcytosis was not affected. In this condition, before addition to the cell system, the ELDKWA peptide was incubated alone (without Abs) at 4°C overnight followed by dialysis with a cutoff at 10 kDa to remove the free peptide. Incubation with the ELDKWA scrambled peptide did not alter the neutralization efficiency, indicating the specificity of the ELDKWA peptide sequence in Ab-mediated inhibition of transcytosis. The ability of ELDKWA to more strongly diminish transcytosis inhibition by S-IgA than by IgG (55 vs 5%, respectively) is consistent with the ability of S-IgA to bind ELDKWA more strongly than IgG (Fig. 1Go, b and d). The amount of colostrum 48 and cervicovaginal fluids were insufficient for analysis in the transcytosis assay.

Expressing HIV-1 transcytosis inhibition as a function of the specific activity of the Ig for ELDKWA (Fig. 1Go) revealed that the capacity to neutralize transcytosis and the involvement of ELDKWA in this process is much more efficient for S-IgA than IgG. Prevention of neutralization by ELDKWA was affected neither by the HIV-1 subtype used to infect PBMCs nor by the origin of the epithelial cell line used in the transcytosis assay. These results reinforce the concept that ELDKWA is a crucial epitope in HIV-1 infection. Indeed, it is the minimal hexapeptide recognized by the transcytosis-neutralizing clostral S-IgA (present studies), dimeric IgA (11), and neutralizing 2F5 IgG (26).

Both HIV-1 envelope gp41 and gp120 subunits interact with epithelial cell Gal Cer

The ability of anti-ELDKWA Abs to inhibit HIV-1 transcytosis suggested that the hexapeptide epitope of gp41 participates in the interaction between HIV-1 and its epithelial cell target. The glycosphingolipid Gal Cer has been shown to act as the gp120 receptor on epithelial cells (14, 15). We have previously shown that this interaction likely plays a role in transcytosis because Abs to both anti-gp120 and anti-Gal Cer completely blocked HIV-1 transcytosis (10). Based on structural and thermodynamic considerations (A. A. and M. B., manuscript in preparation), we investigated whether gp41 also interacts with Gal Cer and whether this interaction involves hexapeptide ELDKWA.

First, as shown morphologically in Fig. 3Go, recombinant gp120 as well as gp41 bind to the apical surface of a tight epithelial cell barrier. Both interactions were antagonized by monoclonal anti-Gal Cer Abs. In contrast, only the gp41 interaction with the apical epithelial surface was inhibited by a 100 molar excess of the ELDKWA peptide, whereas the binding of gp120 was not altered by this peptide. That gp41 bound specifically to Gal Cer via ELDKWA is also supported by the inability of an irrelevant or an ELDKWA scrambled peptide to impair this interaction and the lack of binding of control human IgA to the apical surface of the cell (data not shown).



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  		FIGURE 3. gp41 binds to epithelial apical surface in a ELDKWA epitope-dependent manner, and epithelial Gal Cer acts as gp41 epithelial receptor. Recombinant gp120 (1, 2, 3) or gp41 (4, 5, 6) glycoproteins were introduced into the apical medium bathing the apical HT-29 epithelial cell monolayer surface in the presence of a 100 molar excess of the ELDKWA peptide (2 and 5) or a monoclonal anti-Gal Cer Ab (3 and 6). Binding of recombinant HIV-1 envelope glycoproteins was detected by indirect immunofluorescence by confocal microscopy. gp41 and gp120 bind specifically to the apical epithelial cell surface on Gal Cer, but only the binding of gp41 was displaced by the ELDKWA peptide. Bar, 10 µm.

 
Second, by using a ligand blotting approach, we showed that recombinant gp41, similar to gp120, binds Gal Cer immobilized on a NC support (Fig. 4Goa). No binding to other glycolipids, such as the ganglioside GM1, or a phospholipid, such as PC, was detected (Fig. 4Goa). As a control for specificity, we also showed that CT-B bound only its known receptor GM1 (27) and not Gal Cer or PC (Fig. 4Goa).

Third, a 100 molar excess of the ELDKWA peptide completely inhibited the interaction of gp41 with Gal Cer (Fig. 4Goa, arrow) but not the interaction between gp120 and Gal Cer. ELDKWA scrambled peptide did not inhibit binding of gp41 to Gal Cer, and the binding of CT-B to GM1 was not sensitive to the presence of the ELDKWA peptide, further indicating that gp41 binding with Gal Cer involved ELDKWA.

We next compared the capacity of S-IgA and its paired IgG to neutralize HIV-1 transcytosis (Fig. 2Go) with their capacity to inhibit the binding of gp41 to Gal Cer. In this experiment, ligand blotting was performed in the presence of sample 63 or 64 S-IgA or IgG. Igs from these patients were chosen because a relatively large quantity of material was available, S-IgA bound ELDKWA substantially better than its paired IgG (ratio of ~20, see Fig. 1Gob), and the S-IgA was highly effective in neutralizing HIV-1 transcytosis (>84%, see Fig. 2Go). Results obtained with samples from patients 63 and 64 were identical, and only those obtained with sample from patient 63 are shown on Fig. 4Gob. As shown in Fig. 4Gob, S-IgA alone efficiently blocked gp41 binding to Gal Cer (Fig. 4Gob, arrow) but not gp120 binding to Gal Cer. Additionally, Gal Cer binding of both gp41 and gp120 was unaffected by paired clostral IgG. Finally, we addressed whether the capacity of S-IgA to inhibit gp41 binding to Gal Cer could be ascribed to the efficiency of S-IgA binding to ELDKWA (Fig. 1Go). S-IgA (63 or 64) or paired IgG were preincubated with ELDKWA, and unbound peptide was removed by dialysis, as in Fig. 2Go. The Igs were assayed for inhibiting gp41 or gp120 binding to Gal Cer. S-IgA, not matched IgG, preincubated with ELDKWA had lost its capacity to inhibit gp41 binding to Gal Cer (Fig. 4Gob). As additional control for specificity, an ELDKWA scrambled irrelevant peptide was used and had no effect (data not shown). These results indicate that ELDKWA is a gp41 epitope functionally involved in HIV-1 binding to and transcytosis across epithelial cells and that ELDKWA is the epitope used by the S-IgA for neutralizing HIV-1 transcytosis.

Disruption of the organization of raft microdomains containing Gal Cer inhibits HIV-1 transcytosis

Glycolipids (mainly Gal Cer) and cholesterol form dynamic microdomains functioning at the apical epithelial membrane (16, 28). These microdomains create floating rafts in otherwise phospholipid-rich membrane environments and are involved in membrane trafficking to and from the apical membrane, including apical endocytosis (17, 20) and transcytosis (19, 20). These transient microdomains have been shown to be stabilized by cholesterol (29), and depletion of cholesterol from cell membrane destabilized these raft microdomains (29), therefore impairing their functional activity. To investigate the functional role of Gal Cer containing raft microdomains in HIV-1 transcytosis, the raft organization was disrupted by cholesterol depletion using m{beta}CD.

We first established the optimal conditions for m{beta}CD disruption of raft microdomains without affecting the integrity and tightness of the epithelial barrier (see Materials and Methods). Treatment for 1 h at 37°C with 1–10 mM m{beta}CD, concentrations usually used to disrupted the domain (29), did not alter the TER of the epithelial barrier (Fig. 5Go). The drug was then removed and HIV-1 transcytosis was assessed as described above. As shown in Fig. 5Go, m{beta}CD inhibited HIV-1 transcytosis in a concentration-dependent manner with almost total inhibition at 10 mM. m{beta}CD pretreatment of epithelial monolayers did not modify the apical production by HIV-1+-PBMCs of HIV-1 presented for transcytosis (data not shown), and therefore could not account for the inhibition of transcytosis. The effect of m{beta}CD on transcytosis was reversible when the drug was removed and epithelial cells were allowed to replenish their cholesterol content for 12 h prior the onset of the transcytosis assay (data not shown). These data suggest that HIV-1 transcytosis required the interaction of HIV-1 envelope glycoprotein subunits with the apical epithelial Gal Cer as well as the functional organization of Gal Cer in raft microdomains stabilized by cholesterol.



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  		FIGURE 5. Inhibition of HIV-1 transcytosis by m{beta}CD that disrupt Gal Cer containing raft microdomains. Epithelial cell monolayers were preincubated for 1 h with increasing amounts of m{beta}CD as indicated. After washing the drug out, HIV-1-infected cells were added apically, and transcytosis was measured as in Fig. 2Go. The transcytosis results (closed bar) are expressed as the percentage of the transcytosis inhibition compared with transcytosis in the absence of drug. TER was measured at the end of the m{beta}CD treatment prior the onset (•) and at the end ({circ}) of transcytosis and is expressed as the percentage of inhibition of the TER compared with standard condition of transcytosis in the absence of drug. Bars correspond to mean ± SEM of data pooled from at least three independent experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mucosal surfaces are the major routes by which HIV-1 enters the host in the majority of HIV-1 infection, and epithelial cells are one of the first cells encountered by the virus during mucosal transmission. The HIV-1 envelope plays an essential role in the initial interaction between HIV-1 and its target cells and, consequently, is a potential target for future vaccines. Because HIV-1 envelope glycoproteins vary, conserved sequences are more suitable epitopes for neutralizing Abs. Here we characterize a novel conserved region, ELDKWA, on gp41 involved in the interaction between HIV-1 and CD4-negative epithelial cells. Two recent studies have emphasized the importance of the ELDKWA peptide in mucosal transmission of HIV-1, because passive transfer of IgG, including Abs to ELDKWA, completely inhibited SIV and chimeric HIV-1/SIV mucosal transmission in vivo (30, 31). We show that ELDKWA is a critical region of gp41, which is functionally targeted by neutralizing S-IgA from HIV-1-seropositive subjects. Taken together, these results predict that induction of mucosal IgA directed at the ELDKWA epitope may protect against HIV-1 mucosal transmission at mucosal sites. Better understanding of the exact nature of this peptide region of gp41 could help design a potential target for a mucosal vaccine.

The HIV-1 envelope is organized as an oligomeric structure comprising multimers of gp120 and gp41 molecules, referred to as "spikes," in which gp120 interacts with and masks a large part of gp41 (32, 33, 34). Our results, combined with those of others (14, 15), indicate that both subunits interact with the epithelial target cell by binding Gal Cer at the apical surface of the epithelial cells. This agrees with the proposed physical organization of the spike in which both the Gal Cer binding sites, the one on gp120 (15, 32) and the one described here involving ELDKWA on the external portion of the gp41 C-terminal helix (26), are exposed and therefore accessible to neutralizing Abs.

The lipid composition of the apical epithelial membrane exhibits unique features. Its external leaflet is highly enriched in glycosphingolipids (35, 36). Gal Cer, a monohexosylceramide, is a glycosphingolipid characteristic of the apical membrane epithelial cell. Transient lateral assemblies of glycosphingolipids stabilized by cholesterol, referred to as raft microdomains (18, 37, 38), function in epithelial cells as platforms for trafficking toward the apical membrane as well as in apical endocytosis (17, 20). Raft microdomains are also involved in transcytosis across endothelial cells (39). Apical lipid rafts invaginate forming caveolar structures that contain the cholesterol-binding protein caveolin (40, 41). Such dynamic structures are endocytosed allowing endocytosis and transcytosis (20). Cholesterol depletion completely abolishes the formation of apical caveolae and blocks internalization and transcytosis of clustered raft proteins (20), indicating the stabilizing role of cholesterol in raft structure and the highly dynamic nature of raft microdomains.

Here we have shown that the interaction between HIV-1, and more specifically ELDKWA, on gp41 with epithelial cells is dependent on the raft organization on the apical epithelial membrane. Our studies reinforce the concept that HIV-1 is endocytosed at the apical surface and then transcytosed to the basolateral surface. First, HIV-1 envelope glycoprotein subunits bind specifically to Gal Cer, an essential component of raft structure and dynamics. Second, disruption of the organization of raft microdomains completely blocks transepithelial transcytosis of HIV-1. HIV-1 may enter epithelial cells via caveolar structures. The distance between the two Gal Cer binding sites on gp120 and gp41, estimated to be <7 nm (32, 34), is in good agreement with the proposed size of a stabilized raft microdomain having an estimated size between 3 and 50 nm in diameter (29, 37, 42). Therefore, the interaction of one spike of gp120-gp41 multimers at the surface of the virion with one domain of the target epithelial cell membrane could be a cooperative one. The primary contact of the gp120 part of the spike with a Gal Cer microdomain would stabilize this raft domain, allowing a favorable orientation and interaction of the gp41 exposed peptide within the same space. However, the involvement of clathrin-coated structures cannot be entirely excluded yet, as cholesterol depletion has also been shown to affect clathrin-dependent endocytosis in epithelial cells, although to a much lesser extent (20).

We and others have previously shown that transcytosis of the virus budding from HIV-1-infected mononuclear cells is dependent on the contact between the HIV-1-infected cell and the apical epithelial cell surface (10, 11, 12). Transcytosis, but not the viral budding event, is inhibited by Arg-Gly-Asp added to the apical surface. Arg-Gly-Asp peptides broadly inhibit cell-cell contact mediated by integrin/disintegrin interactions (M.B., unpublished data). Because epithelial cells are known to express adhesion molecules like ICAM-1 apically, these results suggest that the HIV-1 infection of mononuclear cells, which up-regulates surface expression of integrins like LFA-1 (43), interact with the target epithelial cell via integrin-disintegrin interactions. Such interactions would circumscribe a microenvironment between the two cells, which resembles the recently described immunological synapse (13, 44, 45). Actin is required for polarized HIV-1 budding from HIV-1-infected cells at the epithelial cell surface (46). Similarly, the dynamics of actin filaments polymerization, induced upon cell-cell contact and mediated by integrins, play an important role in the formation of the immunological synapse. Actin filaments organize the synapse into distinct supramolecular activation clusters that favor the initiation of signal transduction cascades. The actin scaffold would recruit or stabilize specialized membrane domains enriched in glycolipids (13, 47). The interaction of HIV-1-infected mononuclear cells with epithelial cell inducing HIV production and transcytosis may be similar to the formation of immunological synapse for T cell activation. This adhesion mechanism would overcome the resistance to cell-cell contact posed by the negatively charged glycocalyx present also on these cells. In summary, as described for the immunological synapse (13), adhesion of HIV-1-infected cells to the apical epithelial cell surface may induce a rearrangement of the epithelial actin cytoskeleton that, in turn, would facilitate recruitment and stabilization of the glycolipid raft microdomains by the HIV-1 envelope and initiate the cascade of events leading to endocytosis of the virus.

Several gp120 and gp41 epitopes have been shown to be the target of neutralizing Abs. 2F5, a monoclonal IgG purified from peripheral B cells of an HIV-1-seropositive patient (24, 48) that recognizes the epitope ELDKWA on gp41, neutralizes infection by a large array of HIV-1 primary isolates both in vitro and in vivo by passive immunization (30, 31). However, 2F5-like Abs that possess both immunochemical specificity for ELDKWA and neutralization activity are seldom present in the sera of HIV-1-infected patients, and numerous attempts to produce such neutralizing IgG in vitro or in animals in vivo have failed (49). In contrast, our results show that S-IgA, but not IgG, specific for ELDKWA and possessing neutralizing activity for HIV-1 transcytosis is usually present in secretions from HIV-1-seropositive patients. Thus, ELDKWA appears to be immunodominant for a mucosal but not for a systemic immune response.

Why are neutralizing systemic IgG not generated against ELDKWA, whereas in the context of TM-gp41 this peptide appears immunogenic for humoral mucosal immunity and elicits S-IgAs that neutralize transcytosis of HIV? Several non-mutually exclusive parameters can be invoked.

First, the structure of neutralizing Abs: the multimeric structure of S-IgA, as compared with monomeric IgG, most likely plays a role in the affinity/avidity of the Ab for the target (50) and, therefore, for the neutralizing function. HIV-neutralizing IgGs do not have a remarkably rapid mutational rate nor do they reveal striking pressure for replacement mutations in V region framework regions and complementary-determining regions. However, both the neutralizing 2F5 and 2G12 IgGs that recognize, respectively, the ELDKWA peptide and a region on SU-gp120 carry an unusual heavy chain complementary-determining region 3 that might mediate their respective functions and probably also account for their rare occurrence in patient sera (49). The existence of such specific structures in neutralizing S-IgA remains to be established.

Second, the compartmentalized induction of mucosal vs systemic humoral immunity (51, 52), and within the mucosal compartment itself, as has been recently demonstrated (53, 54, 55). However, mechanisms responsible for compartmentalization remain unclear.

Third, the tolerance induced by self-peptide (56): self-peptide could originate from a soluble protein like the human complement factor H present at high concentration in the plasma, which has been shown to bind to TM-gp41 at a site overlapping the 2F5 epitope (57).

Fourth, the structure of the epitope that elicits neutralizing Abs: ELDKWA is the minimal hexapeptide recognized by neutralizing 2F5 IgG (26) by clostral S-IgA (present studies) and by dimeric IgA as we have shown earlier (11). However, injection of this hexapeptide in animal models never leads to the induction of neutralizing Abs, whatever the site of induction and the class of Ab (IgG or IgA) induced. This indicates that either the immunogenically functional epitope is larger than the hexapeptide and/or that the conformational structure of this peptide within TM-gp41 is of great importance for its immunogenicity. Furthermore, even if the ELDKWA peptide has been found identically conserved in >85% of HIV of the main viral subtypes (A-H), some rare isolates harbor in this region a partial conservation with the leucine (L) and the tryptophan (W), as is also the case for HIV-2 (EHO) and SIV (mac239) (58).This suggests that not only the sequence but also the structure of the region encompassing ELDKWA is of importance and could define a mimotope, essential for HIV transcytosis via Gal Cer binding and induction of humoral immunity. It is striking that the overlapping peptide DP178/T20 (aa 638–673) is able to neutralize infection of cells in vitro by primary HIV isolates, and has been recently shown significantly to reduce the level of circulating HIV when administered to patients for 14 days (25). Indeed, DP178/T20 has been shown to adopt a helical structure and to assemble in tri- or tetrameric oligomers. Furthermore, the amino acid sequence 650–684 of TM-gp41 contains a stack of aromatic hydrophobic residues (W and F) that extend the DP178 sequence at its C-terminal end up to the transmembrane region (aa 684) (59). These tryptophans have been very recently found to be essential for HIV interaction with its target, at a post binding but pre-fusion step (58) as is the case here, because HIV do not fuse with the epithelial cell membrane during transcytosis (10). This suggests that this stack contributes to the structure of the sequence starting at the ELDKWA peptide and extending N terminally to the charged polar amino acid up to the reported Ca2+ binding sites (60) by organizing the covering water surface layer. It would create a water layer between the peptide and the cell membrane that in turn would facilitate charge transfer processes (A. A. and M. B, manuscript in preparation) being at the origin of the signal for endocytosis (61).

The data presented here suggest that the ELDKWA hexapeptide of gp41 is a potential candidate Ag for a mucosal HIV-1 vaccine. First, the peptide is a conserved epitope. Second, ELDKWA is involved in the binding of gp41 to its epithelial receptor Gal Cer and is functionally involved in HIV-1 translocation across a tight epithelial barrier. Third, ELDKWA is an immunodominant Ag in the mucosal humoral response in seropositive patients. Fourth, S-IgA developed against the hexapeptide functionally blocks transcytosis, likely by interfering with the binding of gp41 via ELDKWA to epithelial Gal Cer. Fifth, as part of the DP178/T20 peptide that inhibits HIV-1 infection in cell culture experiments, ELDKWA is reported to have an antiviral effect in a phase I clinical trial in humans (25). Finally, the peptide is recognized by the 2F5 IgG that recently was shown to participate in the protection of macaque against SIV or chimeric HIV-1/SIV mucosal infection (30, 31) when used in a passive immunization protocol. Although the precise structure of the ELDKWA containing peptide that elicits in vivo such neutralizing S-IgA has to be determined, the information presented here indicates the critical role the ELDKWA plays in the interaction of HIV-1 envelope glycoproteins and epithelial cells, the first step in epithelial cell translocation of HIV-1.


   Acknowledgments
 
We thank J. M. Reynes from Pasteur Institute, Pnom Phen, Cambodia for providing the cervicovaginal samples, H. Hocini for clostral Igs purification and help with ELISA analysis, G. Pancino, T. Purtscher, and H. Katinger for peptides and the 2F5 mAb, Dr. Labert from PM s&v for the gp160 and C. Desgranges for IgG anti-HIV-1 envelope subunits, F. Russo-Marie and I. Schwarz-Cornil for fruitful discussions and encouragement throughout this work, and I. Schwarz-Cornil, P. D. Smith, S. Moss, and B. Weksler for critical reading and English editing of the manuscript.


   Footnotes
 
1 This work was supported by Agence Nationale de Recherches sur le SIDA funds (to M.B.) and Paris V University funds (to A.A.). P.I. was a recipient of a SIDAction fellowship; E.B. was supported by SIDAction. Back

2 Address correspondence and reprint requests to Dr. Morgane Bomsel, Institut Cochin de Genetique Moleculaire, 22 rue Mechain, 75014 Paris, France. Back

3 Abbreviations used in this paper: Gal Cer, glycosphingolipid galactosyl ceramide; HIV-1+ PBMC, PBMC infected with HIV-1 primary isolates; m{beta}CD; methyl-{beta}-cyclodextrin; TER, transepithelial resistance; NC, nitrocellulose; CT-B, the B subunit of cholera toxin; PC, phosphatidylcholine. Back

Received for publication December 26, 2000. Accepted for publication March 14, 2001.


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 Discussion
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