Mucosal and Plasma IgA from HIV-1-Exposed Uninfected Individuals Inhibit HIV-1 Transcytosis Across Human Epithelial Cells This information is current as of September 30, 2021. Claudia Devito, Kristina Broliden, Rupert Kaul, Lennart Svensson, Kari Johansen, Peter Kiama, Joshua Kimani, Lucia Lopalco, Stefania Piconi, Job J. Bwayo, Francis Plummer, Mario Clerici and Jorma Hinkula

J Immunol 2000; 165:5170-5176; ; Downloaded from doi: 10.4049/jimmunol.165.9.5170 http://www.jimmunol.org/content/165/9/5170 http://www.jimmunol.org/ References This article cites 39 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/165/9/5170.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Mucosal and Plasma IgA from HIV-1-Exposed Uninfected Individuals Inhibit HIV-1 Transcytosis Across Human Epithelial Cells1

Claudia Devito,* Kristina Broliden,2* Rupert Kaul,† Lennart Svensson,‡ Kari Johansen,‡ Peter Kiama,† Joshua Kimani,† Lucia Lopalco,§ Stefania Piconi,¶ Job J. Bwayo,† Francis Plummer,†ʈ Mario Clerici,3 and Jorma Hinkula‡

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial layer. A Transwell system was established to model HIV-1 infection across the human mucosal . The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal Downloaded from model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates http://www.jimmunol.org/ tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals. The Journal of Immunology, 2000, 165: 5170–5176.

ore than 90% of global HIV-1 transmission occurs pathogens. While innate immune factors and cells constitute the across a mucosal surface (1, 2). This may be the gen- first line of defense against invading micro-organisms, Ag-specific M ital tract mucosa, as in the case of heterosexual trans- humoral and cell-mediated responses are generally required for a

mission of HIV-1, or the oropharyngeal mucosa, as in the case of partial or full protection from infection (4). by guest on September 30, 2021 breast milk transmission. Although transmission across intact mu- Some individuals in high risk groups appear to resist HIV in- cosal surfaces can occur, surface abrasions and breaks in the in- fection despite multiple repeated exposure to the virus (5–7). tegrity of the mucosal epithelium increase the risk of infection. These highly exposed, persistently seronegative (HEPS)4 individ- Sexually transmitted diseases and other factors amplifying inflam- uals remain HIV IgG negative as tested by Western blotting and matory T cell populations in regional mucosal sites also exacerbate lack detectable HIV RNA and HIV DNA as measured by PCR. HIV transmission (3). The mucosal immune system protects the Furthermore, they have no clinical or laboratory signs of immu- epithelium and underlying tissues and organs from environmental nodeficiency. It has been suggested that HIV-specific Th and CTL, ␤ chemokines, and a homozygous deletion of the CCR5 receptor confer partial protection against infection in these individuals (5– *Department of Clinical Virology, Karolinska Institute, Huddinge University Hospi- 9). Recently, we reported that they also have mucosal HIV-specific tal, Stockholm, Sweden; †Department of Medical Microbiology, University of ‡ IgA (7, 10) and that these Abs could neutralize primary HIV-1 Nairobi, Nairobi, Kenya; Department of Clinical Virology, Swedish Institute for 5 Infectious Disease Control, Microbiology and Tumor Biology Center, Karolinska isolates in an assay using PBMCs as target cells (11). Institute, Stockholm, Sweden; §Immunobiology of HIV Unit, San Raffaele Scientific In the present report the functional properties of mucosal (cer- Institute, Milan, Italy; ¶Division of Infectious Diseases, L. Sacco Hospital, Disp-Lita Viaeba, Milan, Italy; and ʈDepartment of Medical Microbiology, University of Mani- vicovaginal fluid and saliva) and systemic (plasma) IgA Abs from toba, Winnipeg, Canada HEPS individuals were further explored in a model of HIV-1 Received for publication March 27, 2000. Accepted for publication August 2, 2000. transcytosis across a tight human epithelial cell layer. Polarized The costs of publication of this article were defrayed in part by the payment of page epithelial cells have a plasma membrane that is separated into charges. This article must therefore be hereby marked advertisement in accordance clearly distinct domains by tight junctions: the apical domain, with 18 U.S.C. Section 1734 solely to indicate this fact. which faces the lumen, and the basolateral domain, which faces the 1 This work was supported by grants from the European Community “Concerted serosal side and the internal milieu (12). Transcytosis is the char- Action BMH4-CT97-2055” (to M.C. and K.B.); The Swedish Physicians Against AIDS Research Foundation (to K.B.); Instituto Superiore di Sanita`, X Progetto AIDS acteristic pathway of membrane trafficking, allowing selective and Grant 40.A.0.58 (to L.L.) and Grant 9403-34 (to M.C. and S.P.); the Medical Re- rapid from the apical to the basolateral pole search Council of Canada (to R.K.; fellowship to F.P.; Senior Scientist Award GR13301 to F.P.); Swedish Medical Research Council Grant 06X-13030 (to J.H.); and Rockefeller Foundation Grant RF96034 (to F.P.). 2 Address correspondence and reprint requests to Dr. Kristina Broliden, Department 4 Abbreviations used in this paper: HEPS, highly exposed, persistently seronegative; of Clinical Virology, F68, Karolinska Institute, Huddinge University Hospital, S-141 CVF, cervicovaginal fluid; NSI, nonsyncytium-inducing; SI, syncytium-inducing. 86 Stockholm, Sweden. E-mail address: [email protected] 5 C. Devito, J. Hinkula, R. Kaul, L. Lopalco, C. Barass, S. Piconi, D. Trabattoni, J. J. 3 Current address: Chair of Immunology, University of Milan, Via GB Grassi 74, Bwayo, F. Plummer, M. Clerici, et al. Mucosal and plasma IgA from HIV-exposed 20157 Milan, Italy. seronegative individuals neutralize primary HIV-1 isolates. Submitted for publication.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 5171 of this epithelium (12, 13). In elegant studies, Bomsel et al. (14) D-galactose (pH 7.5) to the beads and was incubated overnight at room have shown that HIV-1 particles were internalized by the epithe- temperature for at least 18 h. The eluate was absorbed with 100 ␮lof lium after contact of HIV-1-infected cells with the apical surface protein G-Sepharose B beads to remove residual contaminating IgG. Less than 0.1% of IgG was present in the samples after adsorption. The purity of an epithelial cell line. The virus crossed the epithelial tight was further confirmed using PAGE and Western blot analyses (19), and barrier without infecting the epithelium itself, using transcytosis. IgA-depleted fractions contained no HIV-1-neutralizing activity (see Foot- The transcytosed particles could subsequently infect host submu- note 5). An ELISA was used for quantification of purified IgA (20), and cosal mononucleated target cells, indicating a new mechanism for IgA purified from pooled normal human colostrum (Sigma, Life Technol- ogies, Paisley, U.K.) was used as standard to perform the quantification. virus spread in vivo. Furthermore, this transcellular pathway was blocked by anti-HIV envelope protein dIgA or pIgM purified from Transcytosis assay HIV-1-infected individuals (15). We now show that IgA from To measure transcytosis, the cDNA pIgR-transfected human intestinal ep- HEPS individuals is also able to block transcytosis of HIV-1 pri- ithelial cell line CaCo-2 was used (21). The cell cultures were performed mary isolates across a model of the tight epithelial barrier, sug- on Transwell nitrocellulose filters (0.4-␮m pore size filter, Costar, Cam- gesting a novel mechanism by which these individuals may be bridge, MA) in MEM containing 10% inactivated FCS (Life Technologies) protected against mucosal acquisition of HIV-1. during 8–10 days, until the cell lines formed tight monolayer cultures. One milliliter of medium per well was exchanged at the basolateral side, and 0.5 ml/well was exchanged at the apical side every 72 h. The tightness of the Materials and Methods epithelial cells was measured as electrical resistance and was Ͼ400 ohm/ 2 Study subjects cm (range, 420–630) at the start of analysis with apical HIV-1 transmis- sion or basolateral IgA addition or sampling as described previously (22, Female sex workers were enrolled through a dedicated sex worker clinic in 23). Transepithelial electrical resistance was measured with a Millicell the Pumwani area of Nairobi, Kenya (codes 320-1803). Despite behavioral ERS resistance apparatus (Millipore, Bedford, MA). To further control the Downloaded from counseling and condom provision, it is estimated that these women have a tightness of the epithelial cell barrier, passage of recombinant p24 Ag minimum of 64 unprotected sexual exposures to HIV-1/year. Women are (Vector) was measured. classified as HIV-1 resistant if they are seronegative at enrollment and IgA purified from samples from the HEPS or low risk control individ- remain both seronegative and PCR negative during at least 3 years of uals (20–30 ␮g from plasma, 4–18 ␮g from saliva, and 0.3–7.9 ␮g from follow-up while continuing in sex work (16). Sex workers included in the CVF, respectively) was added to the basolateral side of the Transwell present study were enrolled during the 1999 annual clinic resurvey, when chambers. Due to the small amount of mucosal material available from

a standard questionnaire was completed, a physical examination per- each subject, a lower concentration had to be used compared with plasma. http://www.jimmunol.org/ formed, and blood and mucosal samples collected. Fifty microliters of medium was collected from the apical side after 0, Italian exposed uninfected heterosexual partners of HIV-1-infected in- 1, 2, 4, 6, 12, 24, 48, and 72 h, respectively, and analyzed by ELISA for dividuals were also enrolled in the study (codes N13-N30). This cohort of IgA content. This medium was replaced with the same volume of fresh couples, discordant for HIV IgG in serum, has previously been described medium. A human IgM mAb directed against the V3 loop of the HIV-1/ (7). All the HIV-IgG-positive partners were undergoing combined anti- IIIB isolate was used as a control (24). retroviral therapy. No correlations were detected between lack of trans- PBMCs (1 ϫ 105/well) infected with either of the two primary HIV-1 mission and CD4 counts, plasma viral load, or CDC stage of the seropos- isolates were added to the apical side of the chamber. Fifty microliters of itive partners (7). All couples reported regular unprotected vaginal medium was collected from the basolateral side at different time points intercourse during the last 3 years. The study subjects were selected from (0–72 h), and the presence of HIV-1 p24 Ag was measured in a capture the two cohorts on the basis of available mucosal and plasma samples. IgA ELISA as previously described (25). purified from mucosal and plasma specimens from healthy low risk HIV- To measure the capacity of IgA to inhibit transcytosis of HIV-1, the cell by guest on September 30, 2021 1-seronegative Kenyan (n ϭ 7), Italian (n ϭ 10), and Swedish (n ϭ 6) cultures were preincubated with the Abs added to the basolateral side for individuals were used as controls. 6 h at 37°C before addition of the HIV-1-infected PBMCs (1 ϫ 105 in- Ethical approval was obtained from University of Nairobi’s scientific fected cells/well) to the apical side. To analyze where infectious virus had ethical review committee, the H. L. Sacco ethical review committee, and been trapped after incubation at 37°C for 12–14 h, 1 ml each of the baso- from the Karolinska Institute ethical committee, respectively. lateral and apical medium, respectively, was collected and analyzed for p24 Ag content, and medium was collected for HIV-1 isolation. HIV-1 isola- Clinical samples tion was performed by coculture of 1 ml of 106 PHA-stimulated PBMCs in a 24-well plate for 14 days. To analyze the epithelial cells for content of Cervicovaginal fluid (CVF) and plasma were collected as previously de- infectious virus, the apical side of the chambers was washed three times for scribed (10). Saliva was collected by different techniques in the two co- 5 min each time with serum-free medium and once with 1% trypsin-con- horts. For Kenyan female sex workers saliva was collected directly into a ϩ ϩ taining PBS without Ca2 and Mg2 to remove cell surface-bound virus. sterile 50-ml tube and frozen at Ϫ20°C within 2 h, and for heterosexual Adding 1 ml of MEM/10% FCS completed the trypsination procedure. partners discordant for HIV IgG status saliva was collected with the Omni- Cells were spun down, and virus isolation was performed from the cell SAL device (Ferle Products AB, Helsingborg, Sweden), which is designed pellet as described above. to collect resting saliva (17). It consists of an absorbent cotton pad that is Inhibition of transcytosis was calculated as follows: (1 Ϫ (absorbance placed under the tongue, thus collecting saliva from the floor of the mouth. value of the p24 Ag content in sample/absorbance value of the p24 Ag ϫ Virus isolates content without sample)) 100. The two HIV-1 isolates (clade B), a nonsyncytium-inducing (NSI; code Results 6727) and a syncytium-inducing (SI; code 6794) isolate, respectively, were Model system collected from PBMCs of asymptomatic HIV-1-infected patients by cocul- tivation with PHA-stimulated PBMCs from two healthy donors The cul- A Transwell model system was used to assess the ability of IgA to tures were maintained as long as increasing levels of HIV-p24 Ag were inhibit the transcytosis of HIV-1. This system consisted of two detectable in two consecutive determinations. The phenotypic character- chambers separated by a nitrocellulose filter on which a tight ization was determined for each isolate, and supernatants from viral cul- tures were stored at Ϫ80°C (18). monolayer of epithelial cells (CaCo-2) was allowed to grow. The lower chamber, filled with medium, represented the basolateral IgA purification and quantification (mucosal) side to which Abs were added. The upper chamber, IgA was purified with jacalin as previously described with minor modifi- filled with medium, represented the apical (luminal) side to which cations (19). Briefly, 200 ␮l of jacalin/agarose beads (Vector, Burlingame, HIV-1-infected PBMCs were added (Fig. 1). Transepithelial elec- CA) were added to 200 ␮l of sera or 500 ␮l of saliva or CVF diluted in 1 trical resistance was measured to ensure that the membrane was ml of RIPA buffer (2% (v/v) Triton X-100, 150 mM NaCl, 600 mM KCl, tight. The tightness of the epithelial cells was further confirmed by 5 mM disodium EDTA, 3 mM PMSF, and 1 ␮g aprotinin/ml in 10 mM Tris-HCl, pH 7.8). The mixture was shaken for 2 h, and the jacalin/agarose the lack of passage of recombinant p24 Ag. The Ag was added to beads with bound IgA were washed three times with RIPA buffer and three the apical side and could not be detected in the basolateral medium times with PBS. The bound IgA was then eluted by adding 2 ml of 0.8 M in any of the consecutive samples collected during 48 h. In the 5172 IgA FROM EXPOSED UNINFECTED INDIVIDUALS INHIBIT HIV-1 TRANSCYTOSIS

FIGURE 1. The Transwell model for Ab inhibition of HIV-1 transcytosis. The tight epithelial cell barrier divides the well into two separate chambers. In this model the ba- solateral medium represents the mucosal side, and the apical medium represents the luminal side of the epithelial barrier.

absence of a cell layer on the filter, the p24 Ag was detectable in apical side of the Transwell system. When using cell-free virus, the the lower chamber within a few hours (data not shown). efficacy of transcytosis was poor (data not shown). When applying either 1 ϫ 106 or 5 ϫ 105 PBMCs/well (four wells of each con- Transcytosis of purified IgA centration), the electrical resistance decreased in two of the eight To evaluate the efficiency and kinetics of IgA transcytosis, purified wells after 6 h. This reduction in electrical resistance indicates that IgA samples representing HEPS individuals were added to the ba- the tightness of the epithelial cell barrier had been damaged and solateral side of the epithelial cell layer. Altogether, IgA purified from 10 CVF samples and eight plasma samples from the female Downloaded from sex workers and from seven saliva samples from the exposed HIV-1 IgG-seronegative partners were used to evaluate the effi- ciency of IgA transcytosis. When IgA purified from CVF and sa- liva were quantified on the apical side after 12–24 h, 5.5–16.8% (median, 9.2%) and 10.3–13% (median, 10.2%), respectively, of the Abs were recovered. Corresponding values for plasma were http://www.jimmunol.org/ 1–2% (median, 1.7%). The percentage of transcytosed IgA was calculated as the ratio of detectable IgA in the apical medium and the total amount of added IgA in the basolateral medium at a given time point. The kinetics of Ab transcytosis across the cell layer is shown for one of the samples (N18) and, for comparison, a human anti-HIV-1 mAb of the IgM class (Fig. 2). The highest IgA con- centrations were seen at 24 and 48 h, respectively. IgG Abs were not transcytosed across the epithelial cell layer (data not shown). by guest on September 30, 2021 Transcytosis of HIV-1 The efficacy of HIV-1 transcytosis was evaluated by applying cell- free virus or different numbers of HIV-1-infected PBMCs to the

FIGURE 3. Transcytosis of HIV-1. A, Transcytosis of two primary HIV-1 isolates representing different phenotypes (NSI and SI, respectively) across a tight epithelial barrier of CaCo-2 cells. The kinetics of the trans- port of the isolates from the apical to the basolateral side of the cells are FIGURE 2. Transcytosis of human IgA. Transcytosis of human IgA pu- shown. HIV-1 p24 Ag concentrations (picograms per milliliter) were mea- rified from saliva of an HEPS individual (code N18) across a tight epithe- sured on the basolateral side of the cell barrier. B, The basolateral medium lial barrier of CaCo-2 cells. For comparison, an anti-HIV mAb of the collected after 12 h (as described above) was cocultured with fresh human human IgM isotype was included (code M101). The kinetics of the trans- PBMCs for 14 days to determine whether the transcytosed viral particles port of the Abs from the basolateral to the apical side of the cells are remained infectious. HIV-1 p24 Ag concentrations (picograms per milli- shown. IgA and IgM concentrations were compared in the media of the two liter) were measured in the medium of the cocultured PBMCs at different chambers. time points (days). The Journal of Immunology 5173

therefore could not be used for measuring transcytosis. However, when applying 1 ϫ 105 HIV-1-infected PBMCs the electrical re- sistance remained stable (Ͼ400 ohm) for Ͼ36 h in 10 of 10 wells, so this concentration of PBMC was used in all the following ex- periments. Furthermore, electrical resistance was measured in all wells to ensure that the remained intact. If the re- sistance was Ͻ400 ohm/cm2, transcytosis was not evaluated. The efficacy of HIV-1 transcytosis across the epithelial model system was measured by adding PBMCs infected with either of the two HIV-1 primary isolates to the apical side and measuring the HIV p24 Ag concentration in the basolateral medium at different time points. HIV p24 Ag was detected within 1 h (Fig. 3A). These transcytosed viral particles were shown to retain their infectious capacity by coculture of the basolateral medium with fresh PBMCs (medium was collected 12 h post-HIV-1 challenge; Fig. 3B). p24 Ag was never detected in the CaCo2 cells when uninfected PBMCs were tested in a parallel control culture (data not shown).

Inhibition of HIV-1 transcytosis by IgA Downloaded from Having established the efficiency and kinetics of HIV-1 and IgA transcytosis, the ability of IgA to inhibit HIV-1 transcytosis was examined. Purified IgA samples from the saliva of the two HEPS individuals from the Italian discordant couple cohort (N16 and N18) and one Italian low risk uninfected control were added to the

basolateral medium 6 h before addition of HIV-1-infected PBMCs http://www.jimmunol.org/ to the apical medium. The ability of this IgA to block HIV-1 trans- across the mucosal epithelium was examined in our exper- imental model by testing the apical medium, epithelial cells, and basolateral medium for the presence of infectious virus. In case N16, infectious HIV-1 could not be detected in either epithelial cells or basolateral medium, indicating a complete inhibition of HIV-1 (NSI and SI strains) transcytosis by the IgA (Fig. 4A). IgA purified from saliva from case N18 completely inhibited transcy- tosis of the HIV-1 isolate of the SI phenotype, whereas inhibition by guest on September 30, 2021 of the NSI phenotype was less efficient (Fig. 4B). In contrast, IgA purified from the low risk uninfected control individual did not inhibit transcytosis of HIV-1, with high levels of infectious virus detected in the basolateral medium (Fig. 4C). Recombinant gp160 Ag inhibited the blocking capacity by Ͼ80% in two of three IgA samples in the HIV-1 transcytosis assay (Fig. 4D). These observations were extended to include IgA purified from genital tract (CVF), saliva, and plasma of Kenyan HEPS sex work- ers and from saliva of individuals from the Italian discordant cou- ple cohort (Table I). IgA was added to the basolateral side of the epithelial cell layer and was preincubated for 6 h, after which HIV-1-infected PBMCs were added to the apical side. HIV-1 p24 Ag was quantified in the basolateral medium of these experimental wells and compared with the p24 Ag content in control wells without IgA.

FIGURE 4. Inhibition of HIV-1 transcytosis by IgA. Purified IgA sam- ples from saliva of three individuals were added to the basolateral medium 6 h before addition of HIV-1-infected cells to the apical medium. In this way the ability of the Abs to block HIV-1 transcytosis could be measured. Two primary HIV-1 isolates of different phenotypes (NSI and SI) were tested in parallel. A, Subject N16 (HEPS individual). B, Subject N18 (HEPS individual). C, Low risk uninfected control individual. D, Recom- binant gp160 Ag (HTLVIIIB/Lai, clade B) inhibited the effect of IgA pu- rified from saliva of subject 1260. In this example, the results of the HIV-1 isolate (SI) 6794 are shown. Apical medium, cell suspensions, and baso- lateral medium, respectively, were cocultured with fresh human PBMCs for 7 days, after which HIV-1 p24 Ag concentrations (picograms per mil- liliter) were measured (values given in the figures). 5174 IgA FROM EXPOSED UNINFECTED INDIVIDUALS INHIBIT HIV-1 TRANSCYTOSIS

Table I. IgA mediated inhibition of HIV-1 transcytosis across a human both NSI and SI isolates (Table I). No inhibition of HIV-1 trans- epithelial cell layer cytosis was seen in IgA purified from the CVF, saliva, and plasma of low risk uninfected controls (Table I). HIV-1 Primary Isolate (phenotype) Inhibition of Transcytosis (%) Discussion Many HEPS individuals show signs of HIV-specific systemic and Subjecta Sample 6727 (NSI) 6794 (SI) mucosal cellular immune responses as well as the presence of HEPS Individuals HIV-specific IgA Abs (5–7, 10, 11, 26, 27). We recently reported 320 CVF 49 ND that these IgA Abs could neutralize HIV infection of mononuclear 1250 CVF 0 17 cells (11) (see Footnote 5). In the present report we show for the 1700 CVF 65 ND first time that IgA from some of these individuals is able to inhibit 1705 CVF 69 36 1707 CVF 93 32 transcytosis of primary HIV-1 isolates across a human epithelial 1803 CVF 84 69 lining. This activity was seen in IgA purified from both systemic No. of samples Ͼ67% CVF 3/6 1/4 and mucosal compartments in two different cohorts of HEPS in- N13 Saliva 91 0 dividuals. The first cohort consisted of Kenyan HIV-1 IgG-sero- N16 Saliva 100 100 negative female sex workers, and the second cohort consisted of N18 Saliva 66 100 N23 Saliva 92 86 Italian HIV-1 IgG-seronegative heterosexual partners of HIV-1- N28 Saliva 92 36 infected individuals. The IgA-mediated inhibition of HIV-1 trans-

N30 Saliva 0 0 cytosis in these HEPS individuals may contribute to their apparent Downloaded from 320 Saliva 30 22 resistance to HIV infection. 1250 Saliva 34 10 1705 Saliva 19 66 Mucosal IgA able to inhibit HIV-1 epithelial transcytosis was 1785 Saliva 47 ND found in the genital tract of half the Kenyan sex workers tested in No. of samples Ͼ67% Saliva 4/10 3/9 this study. IgA-mediated neutralization of HIV-1 (11) and CD8ϩ 320 Plasma 30 22 lymphocyte responses to HIV-1 CTL epitopes (28) have been pre- 1250 Plasma 0 0

viously described in the genital tract of this cohort. The finding of http://www.jimmunol.org/ 1260 Plasma 100 100 1705 Plasma 5 51 IgA-mediated inhibition of HIV-1 transcytosis therefore suggests 1707 Plasma 93 86 that these highly exposed, seronegative women may have devel- 1803 Plasma 92 69 oped an array of mucosal immune defenses to protect against sex- Ͼ No. of samples 67% Plasma 3/6 3/6 ual HIV-1 acquisition. It is of interest that IgA with the ability to Low-risk uninfected controls 183 CVF 63 37 inhibit HIV-1 transcytosis was not found in the saliva of these sex 184 CVF 30 40 workers, because oral sex is quite uncommon in the sex worker 185 CVF 0 64 cohort (29), while IgA purified from the saliva of seronegative 186 CVF 0 15 subjects in the Italian discordant couple cohort was often able to 187 CVF 29 29 inhibit transcytosis. However, it is also possible that this is a re- by guest on September 30, 2021 188 CVF 5 ND No. of samples Ͼ67% CVF 0/6 0/5 flection of the different methods used to collect saliva specimens in 6 Saliva 53 8 the two cohorts. 7 Saliva 0 3 This study used an experimental system that mimicked natural 9 Saliva 25 59 HIV-1 infection, in which the first steps of infection involve con- 10 Saliva 0 0 13 Saliva 48 0 tact between secretions containing HIV-infected cells or cell-free 17 Saliva 0 0 virus and the mucosal luminal surface (14, 30). The mucosal sur- 20 Saliva 0 0 faces have a covering of either pluristratified epithelial cells (in the No. of samples Ͼ67% Saliva 0/7 0/7 vagina, exocervix, prepuce, and anus) or a simple epithelial mono- S.N Plasma 0 37 layer (in the rectum, endocervix, and intestine). The epithelial cells C.Å Plasma 0 26 L.S Plasma 48 18 are not infected themselves, but due to their polymeric Ig receptor S.S Plasma 0 0 function and polarization they can actively transport (transcytose) M.L Plasma 0 7 molecules such as dimeric IgA and HIV-1 primary isolates to the Å.Å Plasma ND 0 submucosal layer (31). In this study the human intestinal polarized 188 Plasma 26 ND No. of samples Ͼ67% Plasma 0/6 0/6 epithelial cell line CaCo-2 was grown on a filter in a two-chamber (Transwell) culture system (22) in which the investigator had in- a Female sex workers, subjects 320–1803; discordant couples, subjects N13–N30. dependent access to both apical and basolateral medium (14, 15, 32). The culture conditions allowed the cells to form a tight barrier and optimized for polarity development. The upper chamber thus In this way the ability of IgA to inhibit HIV-1 transcytosis was represented the apical (luminal) side, and the lower chamber rep- expressed as a percentage. IgA purified from the CVF of three of resented the basolateral (mucosal) side of the epithelium. In this six Kenyan HEPS sex workers could inhibit at least 67% of HIV-1 model, both IgA Abs and HIV-1 were readily transcytosed across (NSI phenotype) transcytosis. When an HIV-1 virus of the SI phe- the polarized epithelial cell layer by active transportation when notype was used, only one of four of the CVF IgA samples inhib- tested in separate experiments. The HIV-1 particles that were trans- ited transcytosis. Salivary IgA from four of 10 of HEPS individ- cytosed remained infectious, as shown by coculture experiments uals could block at least 67% of the HIV-1 transcytosis of an NSI with mononuclear cells. Within this epithelial model, IgA purified primary isolate, and three of nine could inhibit the HIV-1 trans- from the mucosal and systemic compartments of HEPS individuals cytosis of an SI isolate. This ability of HEPS individuals from the was able to completely or partially inhibit HIV-1 epithelial trans- discordant couple cohort (N13-N30) was limited and was not seen cytosis in about 50% of the IgA fractions purified from CVF, sa- in the saliva of Kenyan sex workers (no. 320-1785). Correspond- liva and plasma samples when tested against at least one of two ing figures for plasma IgA from sex workers were three of six for primary HIV-1 isolates. The main part of the inhibition of HIV-1 The Journal of Immunology 5175 transcytosis by the IgA Abs was shown to be at the intracellular titative viral factors. Although HIV-1 has not been detected in the level rather than on the apical or basolateral side. The frequency exposed individuals, it seems unlikely that induction of these im- with which IgA from HEPS individuals is able to inhibit HIV-1 mune responses could have occurred without at least transient viral epithelial transcytosis must be examined in larger studies, as must replication. Reduction of at-risk sex within the discordant couple the ability of IgA to inhibit transcytosis of differing HIV-1 clades. cohort has been followed by a drop in HIV-specific IgA titers, Although IgA isolated from the mucosa and plasma of half the indicating that HIV-specific immunity in exposed uninfected sub- HEPS subjects studied was unable to inhibit transcytosis, this in- jects could be dependent on a continuous exposure (10) hibition could have been underestimated in the mucosal samples A combination of different immune mechanisms is probably for several reasons. Firstly, a lower mucosal IgA concentration necessary for protection against HIV-1 infection. By defining dif- was used in the assay compared with plasma IgA, and the collec- ferent functional mechanisms of IgA interactions with the virus on tion of mucosal material is less standardized than blood sampling. the mucosal surface we hope to contribute to the development of These factors make comparison of results from mucosal and sys- an efficient vaccine against HIV that would stimulate mucosal hu- temic sites difficult. Furthermore, IgA present in genital secretions moral immunity. may be degraded by IgA proteases, such as those produced by Neisseria gonorrhoeae. For this reason, only sex workers with no Acknowledgments laboratory or clinical evidence of gonorrhea were enrolled in this The human IgM mAb was kindly provided by Dr. Mats Ohlin (University study. Finally, the jacalin used for purification of IgA mainly binds of Lund, Lund, Sweden). We thank Prof. Jan Andersson for valuable com- IgA1, and Abs of the IgA2 subclass may have been missed. These ments, and Marianne Grip for the illustrations. We gratefully acknowledge technical difficulties together with different epitope specificities in Prof. Jean-Pierre Kraehenbuhl (ISREC, Lausanne, Switzerland) for pro- Downloaded from mucosal vs systemic IgA (33) may explain why in some cases the viding the Caco-2 cell line. same individual had divergent IgA responses in the different body fluids. To better elucidate functional IgA responses in the different References body compartments an expanded study would need to include IgA 1. Kresina, T. F., and B. Mathieson. 1999. Human immunodeficiency virus type 1 epitope specificities and subclass distributions as well as quanti- infection, mucosal immunity, and pathogenesis and extramural research programs at the National Institutes of Health. J. Infect. Dis. 179(Suppl. 3):S392. tation of the secretory component (S-IgA). http://www.jimmunol.org/ 2. Overbaugh, J., J. Kreiss, M. Poss, P. Lewis, S. Mostad, G. John, R. 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