Mucosal humoral immunity is mainly mediated by secretory IgA which inhibit HIV-1 transport across epithelium by capturing the transcytosing virions and redirecting them to the serosal pole [1–4]. IgA also inhibit binding of HIV to the glycosphingolipid galactosyl ceramide (Gal–Cer) [5,6] and thereby HIV transcytosis in mucosae [2,4]. Transcytosis, specific for epithelial cells , may allow translocation of HIV-1 across monostratified mucosa that cover endo-cervix, gastro-intestinal tract and rectum . The monostratified transition zone in HIV infection represents one main portal of HIV entry , emphasizing the role of transcytosis in HIV mucosal infection. In pluristratified mucosa infiltrating dendritic cells (DC) and macrophages can also provide a way to systemic HIV-1 spread [10–12]. Although HIV-1 is mainly sexually transmitted, some subjects, despite repeated exposure to HIV-1 by the sexual route, remain seronegative (ESN) . ESN status has been correlated to innate, cellular and humoral immunity such as individuals producing either anti-CCR5 antibodies or HIV-1-specific neutralizing antibodies [14,15]. IgAs from ESN have been shown able to prevent HIV-1 entry at mucosal portals, through transcytosis inhibition  as well as CD4 peripheral blood mononuclear cell (PBMC) infection . Anti-CCR5 antibodies have been also found in long-term non-progressors (LTNP)  although their mechanism of action at mucosal site is unclear.
Material and methods
Anti-CCR5 antibodies were previously found in a subset of ESN and LTNP [14,17–19]. Nine ESN (five males and four females, mean age 35.7 years and mean CD4 counts 932 cells/μl) having anti-CCR5 antibodies were enrolled in this study. Each ESN remained seronegative despite a prolonged history of sexual intercourse without condom use at least twice per week and for at least 2 years. Furthermore, four samples from LTNP with anti-CCR5 antibodies (three males and one female, mean age 44 years and mean CD4 counts 725 cells/μl) [18,20] were also included. The inclusion criteria of LTNP cohort were: (i) certified infection ≥ 7 years; (ii) CD4 T-cells counts ≥ 500 cells/μl; (iii) absence of any antiretroviral therapy; (iv) good overall health. Ten HIV-infected individuals receiving antiretroviral therapy (HIV + HAART), with CD4 counts ≥ 500 cells/μl and no previous AIDS defining diseases, and biological samples from 10 healthy controls (HC) were also included in the study as control populations. All patients were analysed for CCR5-Delta32, CCR2-64I and SDF1-3′A polymorphisms [21–23].
Written informed consent was obtained from all the participants and the study was approved by IRB of San Raffaele Scientific Institute of Milan.
Specimen collection and processing
The cervico-vaginal fluid (CVF) samples were collected by vaginal wash with 7 ml of PBS/EDTA, seminal fluids and saliva were collected in presence of EDTA 10 mM. Samples were centrifuged and stored at −80°C.
Immunoglobulin purification and quantification
CNBr-activated Sepharose 4B columns (Pharmacia, Uppsala, Sweden) coupled with rabbit anti-human total Ig, IgA and IgG (2 mg/ml; Sigma-Aldrich, Milan, Italy) were used to purify total Ig- IgA- and IgG. The column was coupled with rabbit anti-human Ig and then the eluted fractions were further purified on columns coupled with rabbit anti-human IgA and anti-IgG. Eluted IgA- and IgG-fractions, as well as non-Ig fractions, were tested in ELISA. Briefly, microwell plates were coated with dilutions of sera or Ig-containing fractions (up to 1:128 by two-fold dilutions) for 1 h at 37°C. Commercial preparations of human Ig, IgA or IgG (Sigma-Aldrich) were used to generate a calibration curve. After saturation with 1% milk powder (Humana 3, Everswinkel, Germany) in PBS, peroxidase-conjugated goat anti-human IgA or IgG (Sigma-Aldrich) was added and incubated for 30 min at 37°C. The enzymatic reaction was developed with TMB Microwell Peroxidase Substrate System (KPL, Gaithersburg, Maryland, USA) and read at 492 nm.
Synthesis of peptides, preparation of peptide/beads and binding of IgA to peptide-dynabeads
Peptides were synthesized by the solid phase F-moc method  and purified by reverse-phase chromatography . An extra-sequence cysteine was added to peptides #3, and #4, to obtain conformationally cyclic peptides (Table 1).
Coupling of CCR5- and unrelated- peptides (Table 1) to tosyl-activated Dynabeads M280 (Dynal, Oslo, Norway) was obtained following standard procedures.
Binding of IgG/IgA to each CCR5 peptide/bead was obtained by incubating 9 μg of purified IgG or IgA to 9 μg peptide/bead for 1 h at 4°C. Bead-conjugated IgA/IgG were eluted from the beads in 0.5 M acetic acid and dialysed.
Cell lines and antibodies
Glioma U87, CCR5 transfected U87 and 293T cell lines, monoclonal antibodies (SIM4 and 2D7) were obtained through the AIDS Research and Reference Reagent Program. HEC1B and HT29 cell lines were obtained through ATCC. Human CCR5-Specific mAb coupled to FITC were from R&D Systems (Abingdon, Oxon, UK) for clone 180, 182 and control isotype, and from Pharmingen (BD Biosciences, Le Pont de Claix, France) for clone 2D7, IgG2a.
CCR5 binding assay
U87 and CCR5 transfected U87 cells were incubated with Ig-enriched fractions from samples; after 45 min of incubation 125I-sheep anti-human IgG or IgA antibodies-F(ab′)2 (Amersham, Buckinghamshire, UK) was added (final concentration 0.1 nM, 0.2 mCi), and the cells were further incubated for 2 h on ice. Unbound radioactivity was separated by centrifugation on a two-step gradient  in 0.3-ml tubes (Nunc, Roskilde, Denmark) as follows: the lower layer consisted of foetal calf serum (FCS) containing 10% sucrose; the upper layer consisted of 80% silicone (Sigma, St Louis, Missouri, USA) and 20% mineral oil (Sigma-Aldrich). The bound radioactivity in the cell pellets was measured in a gamma counter.
CCR5 detection in epithelial cells
HT29 were saturated with 0.66% fish skin gelatin (Sigma) in PBS, without (surface staining) or with 0.025% saponin (surface + intracellular staining). CCR5-specific or isotype control incubated for 30 min at 37°C. CCR5-specific labelling was analysed by XL2 EPICS flow cytometer (Coulter).
CCR5 internalization assay
CCR5 downregulation was evaluated as previously reported . Briefly, untransfected and CCR5-transfected U87 cell lines, and CD4 T lymphocytes were incubated with affinity-purified anti-CCR5 Ig (200 ng) at 37°C for 48 h as a complete CCR5 downregulation was achieved after 48 h as previously reported . Surface-expressed CCR5 was detected by using an anti-CCR5 mAb (2D7) and a secondary anti-mouse antibody conjugated with FITC . As negative controls a pool of HC and untransfected (U87) cells were used.
Clade B viruses including HIV #36 (R5) and #45 (X4, R5) were obtained as previously described [14,15]. The infectivity (ID50) of each virus was determined on PBMC from one donor as follows: six replicas (150 μl) of fivefold serial dilutions (from 1:5 to 1:3125) of virus were added to six wells of a round-bottomed Microtiter plate (Nunc) containing 2 × 105 resting PBMC in 75 μl of medium, incubated for 2 h, washed and resuspended in RPMI 1640 medium containing phytohaemagglutinin (PHA) (3 μg/ml) and 10 U/ml recombinant interleukin (rIL)-2. HIV-1 p24 antigen (Aalto Bio Reagents Ltd, Dublin, Ireland) was titrated after 5 and 7 days. Fifty percent infectious dose (ID50) titres were defined as the reciprocal of the virus dilution yielding 50% positive wells (Reed-Muench calculation).
SOS pseudoviruses were produced as previously reported . Briefly, pCAGGS plasmid was used to express membrane-bound Env of the primary R5 isolate JR-FL. Pseudoviruses were produced by transfection of 293T cells with pNL4-3.Luc.R-E- and Env-expressing pCAGGS-based plasmids. As negative control VSV-G pseudovirus (Vesicular Stomatitis virus) was used.
For transcytosis assay, 293T cells were transfected by standard technique with 30 μg of HIV-proviral clones. Supernatant p24gag (Aalto Bio Reagents Ltd) was determined 36 h post-transfection.
HIV blocking infection assays
A total of 2 × 105 resting PBMC were added to 75 μl of serial dilutions of serum and/or purified Ig from ESN, LTNP or HC. Each set of assays was correlated with negative control (HC) and with positive controls, SIM4 (anti-CD4 neutralizing mAb) and 2D7 (anti-CCR5 neutralizing mAb). After 48 h incubation (to obtain a complete CCR5 down-regulation), 75 μl of a virus dilution (ID50 adjusted to 20) was added. The cultures were incubated for additional 2 h and then washed and resuspended in medium containing medium containing PHA (3 μg/ml) and 10 U/ml rIL-2. Supernatant p24 was determined on post-infection days 5 and 7, and the analysis was performed when the tissue culture (TC) ID50 level, ranging from 10 to 30, was achieved. The ID50 titres were defined as the reciprocal of the virus dilution in 50% of the positive wells (Reed-Muench calculation). Each value obtained with a specific sample dilution was compared with the mean values from the six corresponding replicates without the addition immunoglobulins and expressed as a percentage of infectivity reduction.
SOS pseudoviruses were used to infect U87 cells. Single-round infections were performed using U87/CD4/CCR5 as target cells. In brief, cells were incubated with different concentrations of antibodies for 48 h (to obtain a complete CCR5 down-regulation), thus SOS pseudoviruses (HIV-R5 and VSV-G) were incubated with U87 cell line for 2 h, the medium was replaced, and appropriate antibody concentrations were again added to the cells. The cultures were then incubated for further 48 h and luciferase activity was measured. The reaction was read by Top Count (Packard, Meriden, Connecticut, USA).
HIV-1 transcytosis was performed as previously described . Briefly, the endometrial cell line HEC-1 or intestinal cell line HT-29 clone 19 cells  were each grown as a tight, polarized monolayer (1 × 106 cells/12-mm diameter filter unit in DMEM glutamax, 20% FCS) for 7/10 days, respectively on a permeable filter support (0.45-μm pore size), forming the interface between two independent chambers, the upper one bathing the apical surface of the epithelial monolayer, and the lower one bathing the basolateral surface. When indicated, purified anti-CCR5-Ig -IgA and -IgG (20 μg/ml), or 2F5 (a neutralizing monoclonal IgG to gp41) or a monoclonal anti-CCR5 antibody 2D7 (10 μg/ml), or IgA or IgG from HC at the indicated concentration were pre-incubated with the apical pole of the epithelial monolayer for 1 or 24 h at 37°C as indicated. Then, HIV-1 infected cells were inoculated to the apical chamber. After 2 h, inhibition of transcytosis by antibody was determined by detection of p24 in the basolateral medium by commercial ELISA (Coulter, Villepinte, France). In some assays, Igs were incubated over night a 4°C with pep#3 or control peptide prior addition to epithelial cells.
Anti-CCR5 antibodies in a subset of ESN and LTNP subjects
HIV-1-neutralizing anti-CCR5 antibodies have been found in a subset of ESN [14,17] and in LTNP . Thus, upon on the availability of the samples, four LTNP and nine ESN subjects having anti-CCR5 antibodies were studied. Negative controls were HIV + HAART-treated patients and HC; these tested negative for anti-CCR5 antibodies .
IgG- and IgA-purified fractions from mucosal secretions and blood were assayed on CCR5-transfected U87 cells. CCR5-specific serum IgG were found in all nine ESN sera, and serum IgA were present in all sera but one (ESN#34) (Fig. 1a; ). Anti-CCR5 IgA were also isolated from six genital secretions (five male, one female). All four sera from LTNP contained CCR5-IgA; all sera but one (LTNP#21) also contained CCR5-IgG. Exclusively Ig fractions were reactive in radio-binding assays, while immunoglobulin-depleted fractions did not bind CCR5-U87 cells (data not shown). Similarly, none of the Ig fractions from HC displayed reactivity (150–850 c.p.m.). Control binding experiments, performed on the untransfected U87 cell line, also gave negative results (200–750 c.p.m.). Moreover, all IgG and IgA fractions were reactive to peptide #3 only. No reactivity was found with the other peptides used in this study (Table 1). CCR5-specific antibodies were then quantified in some samples; the mean was 6.3% of total serum Ig.
All subjects having anti-CCR5 antibodies were screened for other signs of autoimmune antibodies, including antibodies to DNA, extractable nuclear antigens, mitochondria and thyroglobulin and they resulted negative, as already reported [14,18].
Anti-CCR5 IgA block R5-HIV-1 infection
Serum anti-CCR5 Ig in ESN have previously been described to block HIV-1 infectivity by CCR5 downregulation . Moreover, CCR5-specific mucosal IgA have also been identified in the same cohort of ESN . Here we tested serum and mucosal anti-CCR5 IgA in neutralization assays using either PBMC or CCR5 transfected cell lines. PBMC were activated at time of infection as, in these conditions, low level of CCR5 surface-expression is appropriate to set up a CCR5-dependent neutralization assay [14,18]. Two clade B viruses, HIV#40, an R5-tropic virus and HIV#45 an R5, X4-tropic strain, were used. All IgA samples showed a blocking activity to HIV#40, comparable to that of the SIM4 (Fig. 1b). Similarly, anti-CCR5 IgA from LTNP sera reduced infection of HIV#40 (Fig. 1c), while no reduction was found with HIV#45 (data not shown). Block of infectivity was also confirmed in a more sensitive assay. CCR5-specific-salivary IgA (Fig. 1d) and -genital IgA (Fig. 1e) from two LTNP blocked the infectivity of JRFL pseudovirus, while they did not affect infectivity of control pseudovirus (VSV-G) (data not shown). The specificity of anti-CCR5 antibodies was further demonstrated by means of pre-incubation of IgA to either relevant or irrelevant peptide. As shown in Fig. 1d and e, pre-incubation of either salivary or genital IgA with pep#3 but not with an irrelevant peptide showed a decrease in infectivity reduction.
Mucosal immunoglobulins from ESN and LTNP inhibit transcytosis
The mucosal model , occurring equally through epithelial cell lines and human mucosal biopsies , consists of a filter support unit hosting a tightly joined monolayer of epithelial cells. As shown in Fig. 2a, the cell monolayer divides the well into two separated chambers: apical and basolateral. Infected PBMC are applied apically. The contact between infected and epithelial cell forms a synapse  and induces the polarized budding of viral particles . Infectious HIV-1 particles are then transcytosed across the epithelial monolayer before reaching – still infectious – the basolateral chamber of the device mimicking the serosal side of the mucosa . Such basolateral HIV-1 particle did not result from paracellular transport of neither the infected cells nor the cell free-viruses. Furthermore, it did not result from epithelial cell infection as transcytosis occurs from 30 min post-apical contact, time too short to allow viral replication in epithelial cells; furthermore zidovudine-treated epithelial cells still undergo transcytosis . Hence, the level of p24 antigen detected in basolateral chamber after two hours of apical contact between HIV-infected cells and epithelial cells correspond exclusively to the amount of transcytozed virus  during this time and is referred to as 100% transcytosis.
As CCR5 is expressed on the surface of mucosal epithelial cells in vivo as well as on several epithelial cell lines , we verified CCR5 expression on surface of HT29 clone 19 by flow cytometry with mAb recognizing two different epitopes on the second CCR5 extracellular loop (IgG 2D7; and IgG 180 and 182) .
The antibodies bind HT29 either on the surface or intracellularly (Fig. 3a), as it has been shown for the HEC-1 epithelial cell line [32,33] that is capable of HIV transcytosis . We next investigated the activity of CCR5-antibodies on HIV-1 transcytosis. As shown in Fig. 2b, Ig were first applied for 1 h at the apical pole of the epithelial monolayer at various concentrations in transcytosis assays, prior to the addition of infected cells to the apical medium. As a result of specimen availability four IgA samples from ESN sera, three IgA samples from genital fluids, and three IgA samples from LTNP sera were assayed in the mucosa model.
We first investigated whether total Ig from ESN could block in vitro transcytosis. Total Ig from two ESN inhibited transcytosis across HT29 cell monolayer of the B clade HIV-1 primary isolate YU-2, a CCR5 tropic strain but not of a CXCR4 tropic strain, the clade D-HIV-1 NDK (Fig. 3b, c). HC-Ig-pool did not block transcytosis. Furthermore, virus produced at the apical pole of the monolayer during the co-culture in the absence or presence of antibodies was constant (data not shown). Additionally, the production of virus by infected cells only (without epithelial cells), after 2 h of incubation, was not modified by the presence of anti-CCR5 antibodies (data not shown). These results suggest that the anti-CCR5 antibodies inhibited virus release or re-infection within the producer cells, and in turn virus transcytosis in the basolateral medium, as a result of the presence of less virus at the apical surface. We next verified the specificity of anti-CCR5 Ig in the blocking of HIV transcytosis by pre-incubating total Ig from two ESN (#55 and #112) with pep#3 or control peptide prior addition to epithelial cells. Pre-incubation with pep#3 reversed the block induced by the two Ig pool in a specific manner, and control peptide had no effect (Fig. 3b). Additionally, 2D7 had no effect on transcytosis.
Transcytosis-inhibiting IgA recognize a conformational epitope within the first external loop of CCR5
To assess the role of anti-CCR5 antibodies in inhibition of HIV-1-transcytosis, CCR5-specific antibodies were purified on the relevant synthetic pep#3 as previously described . CCR5-specific serum IgA samples from ESN blocked transcytosis of a CCR5 tropic HIV-1 strain YU-2 in a dose-dependent manner (Fig. 4a). Similar results were obtained with another CCR5-HIV-1 strain, JR-CSF (data not shown). As shown in Figs 3 and 4, a decrease in the transcytosis inhibitory activity between total Ig and CCR5-specific IgA, at least for ESN 112 was found. This could be due to the different quantity of IgA versus IgG to CCR5. In fact, it is difficult to establish the role of each component as total Ig contained CCR5-specific Ig of both isotypes. Although in some cases IgA better inhibits HIV, the total amount of IgG is much higher that of IgA, and this could explain why the total amount of Ig better inhibits CCR5-specific IgA. Furthermore, when present together, IgA and IgG could act synergistically. Moreover we cannot exclude the existence of Igs that are not specific to CCR5 but which inhibit HIV transcytosis. Thus, in order to characterize better the role of CCR5-specific antibodies we used affinity purified immunoglobulins on the relevant peptide as shown in Fig. 4.
Because the main function of natural anti-CCR5 antibodies – at least on CD4 T lymphocytes – is downregulation of CCR5 [14,18,29], to characterize the inhibitory activity of CCR5-Ig in inhibiting transcytosis CCR5-IgA samples were pre-incubated with an epithelial cell monolayer for various times at 37°C in a kinetic experiment. Two different conditions were evaluated: a low antibody concentration (7.5 μg/ml) maintained for 24 h, and a shorter exposure to higher antibody concentration (30 μg/ml), for 1 h. IgG and IgA specimens from four seminal fluids and five sera from ESN subjects were tested (Fig. 4b). All IgA samples induced higher inhibition values than respective IgG samples, possibly due to the higher avidity of dimeric IgA. Alternatively, affinity of IgA for CCR5 may be higher than that of IgG. Lower IgA concentrations combined with longer incubation intervals obtained the highest inhibition. Although, in some cases the longer incubation induced a better inhibition of transcytosis, a short 1-h incubation with anti-CCR5 antibody could induce CCR5 recruitment in an intracellular compartment, as revealed by confocal microscopy analysis (not shown). However, the epitope specificity of these blocking CCR5-natural-auto-antibodies could play the major role, as 2D7 recognizes the second extracellular loop of CCR5 and has a very low blocking activity.
Anti-CCR5 antibodies have been described in several cohorts of ESN and in a subset of LTNP, a subpopulation of HIV-1-infected individuals . These antibodies block HIV infection through CCR5 downregulation on T cells [14,18]. However, this paper is the first report that describes the characterization of anti-CCR5 IgA in LTNP and ESN subjects in an in vitro model of human mucosa.
Transcytosis assays, performed in an experimental model of mucosa monolayer, have shown that HIV-1 inhibition could also be mediated by blocking CCR5, although the role of CCR5 at mucosal level is different from that observed in CD4 T lymphocytes .
During transcytosis, HIV-1 does not fuse with the epithelial cells but remains enclosed in transcytotic vesicles and the role of CCR5 in epithelial transport pathway is different from its role of co-receptor on CD4 cells . Accordingly, as the first step of transcytosis, HIV-1 binds to attachment receptors on the epithelial surface including heparan sulfate proteoglycans [5,7,37]. Next, the attached virus interacts with the glycosphingolipid galactosyl-ceramide (or related molecule), and undergoes endocytosis. The role of CCR5 in HIV transcytosis is less clear than in HIV infection. From in vitro experiments on human primary epithelial cells, it has been suggested [34,35] that HIV would bind CCR5 intracellularly in the endosomes. CCR5 would in turn convey HIV through the basal pole of the epithelial monolayer achieving transcytosis.
Here, natural anti-CCR5-antibodies would either bind CCR5 at the epithelial cell surface, allowing CCR5 internalization and reaching CCR5 intracellularly, precluding interaction with HIV and further transcytosis. HIV-1 envelope has recently been shown to interact both with HSPG and CCR5 via the same motif , suggesting that on epithelial cells, HIV-1 would sequentially interact with HSPG prior to interact with CCR5 and achieve transcytosis .
The existence of natural anti-CCR5 antibodies might be explained by the evolution of a ‘permissive’ molecular environment in chronically infected subjects, probably restricted to small local sanctuaries. In fact, LTNP and ESN are able to develop a natural, protective response to HIV. We also used a specific monoclonal antibody to CCR5 (2D7), well characterized to block HIV fusion with target cell prior to infection. The mechanism of action of 2D7 appears clearly different from that demonstrated for natural anti-CCR5 antibodies as 2D7 recognizes a different loop in the extramembrane portion of CCR5, does not induce a long-lasting downregulation of CCR5 on the cell surface of PBMC and does not block transcytosis.
Neutralizing responses also suggest that long-lasting but somewhat controlled exposure to HIV-1 might account for the development and the maintenance of some unusual, protective responses [13,39]. The finding that HIV blocking infectivity is mediated by IgA recognizing the first external loop of the protein might help to explain how a peculiar antigen presentation can be achieved more easily in a circumscribed local environment rather than at systemic level [13,40]. Although the first loop seems to be poorly exposed , it could change its conformational status under specific stimuli, not necessarily HIV related, thus becoming immunogenic. Alternatively, in epithelial cell in the endosome, where HIV is most likely interact with CCR5 to achieve the second leg of the transcytotic pathway [34,35], CCR5 could adopt a non-typical conformation, thus explaining why transcytosis is inhibited by natural antibodies recognizing first loop rather than by antibodies recognizing other CCR5 domains. If the target of natural anti-CCR5 antibodies is indeed CCR5 located in the endosome, it may explain why a higher concentration of natural anti-CCR5 Ig are need to block transcytosis (μg/ml) than to block infection (ng/ml).
Of note is that natural and/or modified CCR5 ligands neutralize HIV by blocking the HIV binding site and/or inducing signaling following short-time kinetics [31,43], while natural antibodies to the first loop of CCR5-mediated HIV infectivity reduction appear to result from the combination of two time-related mechanisms. The shorter one is probably due to steric hindrance, and, more importantly, the second one in which long-lasting CCR5 downregulation is achieved, has been described in LTNP  and in a mouse model . These two mechanisms suggest that a complete and profound block of HIV replication is achieved by anti-CCR5 antibodies. For the first time, we found a mucosal protective response, despite the fact that mucosal tissues throughout the body have been showed to be a primary target for acute HIV-1 replication in humans .
All these findings support the existence of an effective mechanism of natural resistance to HIV-1 mediated by CCR5 downregulation on the mononuclear cell surface or a functional block in epithelial cells that is likely to play a significant role in mucosal protection.
This work was supported by Istituto Superiore di Sanita' grant 40F46 and 40F45 to L.L. and by the Agence Nationale de Recherche sur le SIDA (ANRS) and SIDACTION to M.B. S.G. was supported by la Fondation pour la Recherche Médicale.
We thank D.Burton and J.Binley for providing SOS-pseudovirus. We also thank S. Russo for editorial help.
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