Monoclonal Immunoglobulin A Antibodies Directed against Cholera ...

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Secretory immunoglobulin A (IgA) antibodies directed against cholera toxin (CT) are thought to be ... 23), and recombinant V. cholerae vaccines lacking toxin.
Vol. 61, No. 12

INFECrION AND IMMUNITY, Dec. 1993, p. 5271-5278

0019-9567/93/125271-08$02.00/0

Copyright X 1993, American Society for Microbiology

Monoclonal Immunoglobulin A Antibodies Directed against Cholera Toxin Prevent the Toxin-Induced Chloride Secretory Response and Block Toxin Binding to Intestinal Epithelial Cells In Vitro FELICE M. APTER,1'2t WAYNE I. LENCER,2'3 RICHARD A. FINKELSTEIN,4 JOHN J. MEKALANOS,5 AND MARIAN R. NEUTRA12* GI Cell Biology Laboratory' and Combined Program in Pediatric Gastroenterology and Nutrition, 3 Children's Hospital, and Departments of Pediatrics2 and Microbiology and Molecular Genetics, 5 Harvard Medical School, Boston, Massachusetts 02115, and Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri 652124 Received 26 March 1993/Returned for modification 13 May 1993/Accepted 28 September 1993

Secretory immunoglobulin A (IgA) antibodies directed against cholera toxin (CT) are thought to be important in resistance to oral challenge with virulent Vibrio cholerae, although alternative mechanisms for protection of intestinal epithelia against CT-induced fluid secretion have been proposed. The ability of anti-CT IgA to block the effects of CT on human enterocytes has not been directly tested because of the lack of a well-defined in vitro intestinal epithelial cell system to directly measure toxin action and the limited availability of purified anti-CT IgA antibodies. We have generated hybridomas that produce monoclonal IgA and IgG antibodies directed against CT by fusion of Peyer's patch cells with mouse myeloma cells after oral-systemic immunization of mice with CT and CT B-subunit protein. All of the anti-CT antibodies recognized the B subunit. Three clones (designated anti-CTB IgA-1, IgA-2, and IgA-3) which produced IgA antibodies in dimeric and polymeric forms were selected. Checkerboard immunoblotting demonstrated that IgA-1 recognized an epitope distinct from that recognized by IgA-2 and IgA-3 and that none of the antibodies were directed against the binding site of GM1, the intestinal cell membrane toxin receptor. The protective capacity of these IgAs was tested in vitro with human T84 colon carcinoma cells grown on permeable supports as confluent monolayers of polarized enterocytes. When each anti-CTB IgA was mixed with 10 nM CT and applied to the apical surfaces of T84 cell monolayers, all three IgAs blocked CT-induced Cl- secretion in a dose-dependent manner and completely inhibited binding of rhodamine-labelled CT to apical cell membranes. Thus, monoclonal anti-CTB IgA antibodies are sufficient to protect human enterocytes in vitro against CT binding and action.

cyclase (16) and, in intestinal epithelia, leads to cyclic AMP-dependent Cl- secretion, the primary transport event responsible for secretory diarrhea (5). Binding of CT to enterocyte apical membranes is an essential event in the disease (7, 11, 12). Colonization of the intestine by V. cholerae also evokes both systemic and mucosal immune responses in the host (28). The mucosal response involves production of secretory immunoglobulin A (IgA) antibodies against CT and V. cholerae surface components including the bacterial lipopolysaccharide (LPS) (36). Secretory IgAs are thought to play a role in limiting the duration of the infection and to be important in host resistance to subsequent oral challenge with virulent organisms (2, 15, 20, 21, 36). Although there is evidence that CT binding to isolated microvillus membranes can be blocked by specific antibodies (41), antitoxin immunity in humans is not sufficient to protect against disease (20, 21, 23), and recombinant V. cholerae vaccines lacking toxin genes are protective (22). Furthermore, it has been suggested that other immune and nonimmune mechanisms also play roles in protection against CT in vivo (18, 24). Whether specific anti-CT IgA antibodies can protect intestinal epithelial cells against CT-induced Cl- secretion has not been directly tested. We previously demonstrated that hybridomas secreting dimeric and polymeric IgA antibodies can be readily generated from Peyer's patch cells after oral immunization (29, 39, 40). The number and specificity of the antibodies produced

Vibrio cholerae organisms are motile, uniflagellate, gramnegative bacteria that may cause severe enterotoxin-induced secretory diarrhea. Within the small intestine, V. cholerae expresses a group of coregulated proteins including pilus proteins that promote adherence to mucosal surfaces and colonization of the proximal small intestine and enterotoxin that induces secretion of chloride ions from intestinal epithelial cells (10, 30, 31, 34, 38). Cholera toxin (CT) is the primary agent responsible for the massive secretion of salt and water (reviewed in reference 5) that occurs in the absence of epithelial damage (30). The action of CT on epithelial cells is now partially understood. C0 (86 kDa) consists of five identical B subunits (11.6 kDa each) that form a ring around a single, enzymatically active A subunit (27 kDa). The B subunits are responsible for CT binding to the cell plasma membrane via the ganglioside, GM1 (3, 7, 12). After toxin binding, the A subunit is translocated to the cytoplasmic leaflet of the membrane (reviewed in reference 11) and probably moves by vesicular transport toward the basolateral membrane (9, 19). The A subunit catalyzes the NAD-ribosylation of the regulatory GTPase, Gas (8, 32), which activates adenylate *

Corresponding author.

t Present address: American Association for the Advancement of

Science Diplomacy Fellow at Office of Population, Agency for International Development, Washington, DC 20523.

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by these hybridoma clones provide an indirect indication of the strength and specificity of the mucosal immune response. In addition, the resulting monoclonal IgA reagents can be used as tools to examine the individual contribution of anti-CT IgAs to protection against oral challenge with enteric pathogens or toxins. In this study, we have used such a strategy to obtain anti-CT IgA hybridomas from mice immunized with CT. To directly test the effects of individual anti-CT IgAs on the interaction of CT with intestinal enterocytes, we sought a well-defined, sensitive in vitro system that would allow us to accurately assess both toxin binding and the resultant Cl- secretory response in the absence of nonepithelial cellular and soluble factors. Well-differentiated human intestinal T84 cells in monolayer culture have been used extensively for physiological studies (4) and have recently been defined as a model for the study of CT-induced Cl- secretion (19). When grown on collagen-coated filters, these cells form tight occluding junctions and exhibit vectorial secretion of Cl- in response to a variety of hormones and modulators, including CT. The CT-induced Cl- secretory response of T84 cell monolayers is measured as a dramatic change in short circuit current (Isc) (19). We have now used this model to examine the role of specific anti-CT IgAs in the protection of intestinal epithelial cells. The data show that IgA directed against the B subunit of CT can effectively block CT action on polarized enterocytes in vitro by sterically inhibiting toxin binding to apical cell surfaces. MATERIALS AND METHODS Production and screening of monoclonal IgA antibodies. Three BALB/c mice were perorally immunized on day 0 with 30 ,ug of CT holotoxin from V. cholerae 569B, Inaba serotype (Calbiochem, La Jolla, Calif.; no. 227035). On day 10, mice were perorally boosted with 30 ,ug of CT holotoxin and 100 ,ug of CT B subunit (Calbiochem no. 227039) followed by a boost on day 20 consisting of a 30-,ug peroral dose of CT holotoxin plus an intravenous injection of 100 ,ug of the CT B subunit. On day 25, the mice were sacrificed; Peyer's patches were dissected; and Peyer's patch cells were isolated, pooled, and fused with P3X63/Ag8U.1 mouse myeloma cells as previously described (39). Fusion products were seeded in 96-well flat-bottom plates with a feeder layer of thymocytes isolated from DBA/2 mice. Cultures supernatants were screened by enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated by incubation with a 1-,ug/ml solution of CT in carbonatebicarbonate buffer, pH 9.6, and blocked with 2.5% nonfat dry milk in phosphate-buffered saline (PBS). Hybridoma supematants were applied for 2 h at room temperature or overnight at 4°C. Bound immunoglobulins were detected with anti-mouse IgG-IgA-IgM coupled to horseradish peroxidase (Zymed Laboratories, South San Francisco, Calif.) developed with O-phenylenediamine. Anti-CT monoclonal antibodies were isotyped with kit no. 100-035 from Boehringer Mannheim (Indianapolis, Ind.) according to the manufacturer's instructions. Hybridomas from positive wells were expanded and cloned three times by limiting dilution. The three anti-CT IgA hybridoma cell lines chosen for study were maintained in Serum Free Protein Free Medium (Sigma, St. Louis, Mo.) supplemented with penicillin and streptomycin (GIBCO, Grand Island, N.Y.). Analysis of monoclonal antibodies. All anti-CT IgA and IgG antibodies were tested for subunit specificity by ELISA with isolated CT B subunit or CT A subunit (Calbiochem no.

INFECr. IMMUN.

227037) as immobilized antigen. To obtain a permanent record of the subunit specificity of the three monoclonal antibodies selected for study, CT holotoxin, CT A subunit, CT B subunit, and a control protein, bovine serum albumin (BSA), were used as antigens on a dot blot assay. Proteins were applied to nitrocellulose in a dot blot apparatus (BioRad Laboratories, Richmond, Calif.). The nitrocellulose was removed from the apparatus, blocked with 2.5% nonfat dry milk in PBS, and returned to the apparatus. Monoclonal IgA antibodies were then placed in appropriate wells for 2 h at room temperature. After extensive washing, bound antibodies were visualized with anti-mouse IgA antibodies coupled to horseradish peroxidase (Zymed), developed with 4-chloro-1-napthol. To analyze the molecular forms of monoclonal IgA antibodies produced in hybridoma cell culture, concentrated supernatants were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on a 3 to 15% gradient gel under nonreducing conditions, and proteins were transferred to nitrocellulose. The nitrocellulose blots were incubated with anti-mouse IgA peroxidase (Zymed) and developed with 4-chloro-i-napthol. To determine whether the three monoclonal anti-CTB IgA antibodies recognized identical or distinct epitopes on the CT B subunit, their ability to bind to CT and related enterotoxins was analyzed by checkerboard immunoblotting as previously described (17). The antigens used have been described in detail elsewhere (6). They included the holotoxins and purified B-subunit proteins of cholera enterotoxins (CT) and CT-related heat-labile enterotoxins (LT) from Escherichia coli, as follows: CT-1 and CT-B-1 from V. cholerae 569B (Classical biotype, Inaba serotype); CT-2 and CT-B-2 from V. cholerae 3083 (El Tor biotype, Ogawa serotype); H-LT-B [isolated from H-LT-1, cloned from E. coli H74-114 of human origin into MM294 (pJL10)]; and P-LT-B of the LT from a strain of E. coli of porcine (P) origin. Also tested were a series of chimeric proteins which contain P-LT-B amino acid residues substituted with corresponding H-LT-Bl amino acid residues and designated pDL2, -3, -5, and -7, as previously described (6, 17). To test whether the antibodies specifically recognized the site on C17B which binds the cellular receptor, GM1, the immobilized toxins were pretreated with soluble GM1 at a concentration of 1 ,ug/ml as previously described (17), rinsed, probed with monoclonal IgA antibodies and secondary antibodies, and developed as described above. Concentration and quantitation of monoclonal IgA antibodies. Supernatants from hybridomas grown in Serum Free Protein Free Medium were concentrated in a stirred ultrafiltration cell at 4°C (Amicon, Beverly, Mass.). The concentrate was diluted with PBS and reconcentrated in the ultrafiltration cell. Concentrated IgA in PBS was sterilized by passage through a 0.22-p,m-pore-size p,STAR filter (Costar, Cambridge, Mass.) and stored at 4°C until use. Monoclonal IgA was quantitated by sandwich ELISA with a purified monoclonal IgA (MOPC-315, Sigma) as standard. ELISA plates were coated with anti-mouse IgA antibodies as described for the CT ELISA. Serial dilutions of MOPC-315 IgA and unknown samples of monoclonal anti-CTB IgA were allowed to bind at 4°C overnight. Bound IgA was detected with anti-mouse IgA coupled with horseradish peroxidase (Zymed) and developed with O-phenylenediamine. The reaction products were measured on an ASA 400 ELISA reader (SLT Instruments, Salzburg, Austria). Standard curves were generated (Cricket Graph; Cricket Software,

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Malvern, Pa.), and unknown IgA concentrations were calculated. Measurement of the CT-induced chloride secretory response of T84 cells. Electrophysiologic studies were carried out on confluent T84 cell monolayers (passages 61 and 85) grown on collagen-coated 0.33-mm Transwell inserts (Costar) in 24well culture plates as previously described (19). Monolayers were washed in Hanks buffered salt solution containing (in grams per liter) 0.185 CaCl2, 0.098 MgSO4, 0.4 KCl, 0.06 KH2PO4, 8.0 NaCl, 0.048 Na2HPO4, and 1.0 glucose, to which was added 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.4. A total of 1 ,ug of CT was mixed with either 1, 5, 10, 20, 50, or 100 ,ug of monoclonal IgA antibody in a total volume of 1 ml, and 200 ,ul of the appropriate mixture was applied to the apical surface of each T84 cell monolayer at 37°C. Serosal and mucosal reservoirs of the filter units were interfaced directly with calomel and Ag-AgCl electrodes via 5% agar bridges made with mammalian Ringer's solution consisting of 114 mM NaCl, 5 mM KCl, 1.65 mM Na2HPO4, 0.3 mM NaH2PO4, 25 mM NaHCO3, 1.1 mM MgSO4, and 1.25 mM CaCl2. Transepithelial potentials were measured over time with a dual voltage clamp device (University of Iowa) and 25-,uA current pulses. Short circuit current (Isc) and transepithelial resistance were calculated by Ohm's law as previously described for this cell culture system (4). Morphological assay of CT adherence to 1T4 cells. Rhodamine-labelled CT was prepared as previously described (19). Briefly, 1 mg of lyophilized toxin was reconstituted with 200 pl of 25 mM sodium carbonate, pH 9.2, and immediately incubated at 21°C for 1 h with 0.1 mg of tetramethylrhodamine succidimal ester (Molecular Probes, Eugene, Oreg.) freshly dissolved in N,N-dimethylformamide. The reaction mixture was passed over a Sephadex G-25 (Pharmacia, Uppsala, Sweden) column, and void volumes were collected and pooled. Concentration of labelled toxin was determined byA280 with an extinction coefficient of Al%1 c = 11.42 (25). The molar ratio of rhodamine to CT was determined by comparison of a 0.1% pronase (Boehringer Mannheim) digest of the rhodamine-labelled CT analog (CT-rhodamine) to a 1-mg/ml standard solution of 5- and 6-carboxytetramethylrhodamine in an SLM 8000 fluorimeter (Urbana, Ill.). The CT was labelled at a ratio of 2.4:1 (rhodamine-CT holotoxin) and was functional, with 50% effective dose on T84 cell monolayers identical to that of native CT (19). T84 cell monolayers were grown on permeable supports in 2-cm2 rings as previously described (19), cooled to 4°C, and incubated with 1 ,g of rhodamine-CT per ml with or without 100 ,ug of anti-CT IgA or control IgA in the apical reservoir for 1 h at 4°C. The monolayers were gently washed three times in PBS at 4°C and then fixed in a solution of 2% freshly depolymerized formaldehyde in PBS overnight at 4°C. Cryoprotection of the cells was achieved by incubation of the fixed monolayers in 30% (wt/vol) sucrose in PBS for 4 h. The monolayers on their filter supports were then sliced into strips, stacked together in Cryoform embedding medium (International Equipment Co., Needham, Mass.) in perpendicular orientation on copper blocks, and frozen by immersion in liquid nitrogen. Cryosections (4 ,um) were cut on a cryostat (IEC Minotome), rinsed briefly in PBS, and mounted on glass slides. Coverslips were mounted with Moviol (Air Products and Chemicals Inc., Allentown, Pa.). Sections were viewed on a Zeiss Axiophot microscope, and photographs were taken with Kodak Tmax film (400 ASA).

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RESULTS Monoclonal IgA antibodies recognize CT B subunit. To produce monoclonal IgA antibodies against CT, we immunized mice twice with oral doses of combined cholera holotoxin and isolated B subunit followed by a final boost of oral holotoxin and an intravenous injection of CT B subunit. The fusion of Peyer's patch lymphocytes from three mice resulted in 48 hybridomas secreting anti-CT antibodies, of which 27 belonged to the IgA class. All anti-CT secreting hybridomas identified were shown to be specific for the CT B subunit (CTIB) by their positive reaction on CTB-specific ELISA and lack of reaction on a CT A-subunit (CTA)specific ELISA (data not shown). Three anti-CTB IgA secreting hybridomas were selected for further study on the basis of their sustained growth in culture and high levels of antibodies produced. These monoclonal antibodies were designated anti-CTB IgAl, -2, and -3. Dot blot analysis showed that all three produced monoclonal IgA that recognized the B subunit, but failed to recognize either CT A subunit or a control protein, BSA (Fig. 1A). Anti-CTB IgA-1 and IgA-2 and -3 recognize two distinct epitopes. To determine whether the three monoclonal antiCTB IgA antibodies recognized identical or distinct epitopes on the CT B subunit, their ability to bind CT and related enterotoxins was analyzed by checkerboard immunoblotting. As expected, all three of the monoclonal anti-CTB IgA antibodies bound to the immunogen CT from V. cholerae 569B (Inaba serotype), as well as its isolated B subunit (CT-1, CT-B-1; Fig. 1B). IgA-2 and IgA-3 also bound to CT isolated from V. cholerae 3083 (Ogawa serotype) as well as to its isolated B subunit (CT-2, CT-B-2), while IgA-1 failed to bind to this related toxin. These results suggest that IgA-1 binds an epitope of the CT B subunit distinct from that recognized by IgA-2 and IgA-3. Exposure of the immobilized enterotoxins to 1 p,g of soluble GM1 per ml before exposure to the IgA antibodies did not change the binding pattern of the antibodies on the checkerboard immunoblot, indicating that the epitopes recognized did not include the GM1 binding site and were not affected by possible conformational changes associated with GM1 binding (Fig. 1B). Hybridomas derived from Peyer's patch cells produce IgA in dimeric and polymeric forms. We have previously shown that monoclonal IgA antibodies produced by mucosally derived hybridomas are largely dimeric and polymeric and are recognized by epithelial polymeric immunoglobulin receptors and efficiently transported onto mucosal surfaces (39, 40). To confirm that the monoclonal anti-CTB IgAs in this study were produced in polymeric forms, their molecular weights were analyzed. IgA antibodies were visualized on Western immunoblots of concentrated hybridoma supernatants separated by nonreducing SDS-PAGE. Monoclonal anti-CTB IgA-1, IgA-2, and IgA-3 were all found to be produced in monomeric, dimeric, and polymeric forms (Fig. 1C). Monoclonal anti-CTB IgA antibodies block the action of CT on 184 enterocytes. T84 cell monolayers respond to cyclic AMP agonists with electrogenic C1- secretion and are a well-established model to study the regulation of intestinal salt and water secretion (4). When applied to apical surfaces of T84 cell monolayers, 1 p,g of CT per ml elicits a dramatic cyclic AMP-dependent increase in Isc, and bidirectional flux studies utilizing 22Na and 36C1 have shown that Cl- secretion accounts fully for this increase (19). To examine whether monoclonal anti-CTB IgA-1, IgA-2, or IgA-3 could protect intestinal enterocyte monolayers from

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FIG. 1. Characterization of monoclonal IgA antibodies. (A) Monoclonal anti-CT IgA antibodies recognize the CT B subunit. Dot blot analysis demonstrated that each of three monoclonal anti-CT IgA antibodies recognized cholera holotoxin (CT) as well as its B subunit (CTB) but did not recognize the A subunit (CTA) or a control protein, BSA. A control monoclonal IgA directed against LPS of V. cholerae did not recognize either CT or BSA. (B) Two distinct epitopes of the CT B subunit are recognized by the anti-CTB monoclonal IgAs as demonstrated by checkerboard immunoblotting against CT from different V. cholerae strains and other related enterotoxins. The antigens used included the holotoxins and purified B-subunit proteins of cholera enterotoxins (CT) and CT-related heat-labile enterotoxins (LT) from E. coli CT-1 and CT-B-1 are from V. cholerae 569B (classical biotype, Inaba serotype). CT-2 and CT-B-2 are from V. cholerae 3083 (El Tor biotype, Ogawa serotype). H-LT-B, from H-LT-1, was cloned from E. coli H74-114 of human origin, and P-LT-B is from the LT of a strain of E. coli of porcine origin. pDL2, -3, -5, and -7 are a series of chimeric proteins which contain P-LT-B amino acid residues substituted with corresponding H-LT-Bi amino acid residues as previously described (6, 17). Lanes 1 to 3: each anti-CTB IgA bound the immunogen CT-1 and its isolated B subunit (CT-B-1). Anti-CTB IgA-2 (lane 2) and anti-CTB IgA-3 (lane 3) differed from anti-CT-B-1 (lane 1) in that they also bound CT-2 and its isolated B subunit (CT-B-2). None of the IgA antibodies recognized any other related enterotoxins. Lanes 4 to 6: pretreament of the blot with GM1 did not inhibit binding of any IgA antibody to the recognized toxins and B subunits (lane 4, IgA CT-B-1; lane 5, IgA CT-B-2; lane 6, IgA CT-B-3). (C) Monoclonal anti-CTB IgA is produced in dimeric and polymeric forms. Concentrated hybridoma supernatants were separated by SDSPAGE with a 3 to 15% nonreducing gradient gel and were transferred to nitrocellulose. The blot was probed with anti-mouse IgA coupled to peroxidase and developed. Lane 1, monoclonal anti-V. cholerae LPS IgA, previously shown to produce IgA in dimeric and polymeric forms (37). Lanes 2, 3, and 4, anti-CTB IgA-1, -2, and -3, respectively. IgA was produced by the hybridoma cells as monomers, dimers (lower arrow), and higher polymers (upper arrow).

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FIG. 2. Monoclonal anti-CTB IgA inhibits CT action in a dosedependent manner. For these experiments, 1 ,ug of CT per ml alone, or mixed with varying concentrations of monoclonal IgA antibody, was applied to the apical surfaces of T84 cell monolayers. Transepithelial potential was measured, and Isc was calculated. (A) CT induces a strong Cl- secretory response from T84 monolayers. In these studies, 1 pg of CT per ml alone induced a dramatic change in Isc by 120 min. When the CT was mixed with 100 pg of a control IgA directed against the V. cholerae LPS per ml, there was no inhibition of CT-induced Cl- secretion. (B) Monoclonal anti-CT7B IgA blocks CT action on T84 enterocyte monolayers in a dose-dependent manner. A total of 20 p,g or more of anti-CTB IgA-1 per ml completely inhibited the CT-induced change in Isc measured in T84 cell monolayers, while smaller amounts of IgA decreased the change in Isc. (C) Three anti-CT IgA antibodies show similar dose dependence in inhibition of the CT action. The K1 of each antibody at the peak of the change in Isc, 150 min, is approximately 20 p.g/ml.

the CT-induced Isc response (peak Isc = 1.2 -+- 0.1 .LA/cm2, = 3) (Fig. 2B). Protection by IgA-1 was dose dependent, with complete inhibition at doses of 20 ,ug of IgA per ml or above and no inhibition by 1 ,ug of IgA per ml (peak Isc = 60.2 + 3.5 P,A/cm2, n = 3) (Fig. 2B). Similar results were n

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obtained with anti-CTB IgA-2 and IgA-3 (Fig. 2C). The protective concentration of 20 ,ug of IgA per 1 ,g of CT corresponds to an approximate molar ratio of four to five dimeric IgA antibodies per CT molecule. Anti-CTB IgA antibodies prevent binding of CT to T84 cells. For CT to act on a cell, the toxin must bind via its B subunit to the cell membrane. To examine whether the antibodies blocked CT binding to cell surfaces, we applied 1 ,ug of rhodamine-labelled CT per ml together with 100 ,ug of anti-CTlB IgA antibodies (or 100 ,ug of anti-LPS monoclonal IgA antibodies as a control) to apical reservoirs of T84 cell monolayers at 4°C for 1 h. The monolayers were then rinsed, fixed, cryosectioned, and examined by epifluorescence microscopy. In monolayers incubated with rhodamine-CT mixed with 100 ,ug of control anti-LPS IgA, toxin binding to apical cell surfaces was readily seen (Fig. 3A and B). In contrast, a rhodamine-CT signal was not detected on monolayers exposed to 100 ,ug of anti-CTB IgA antibodies per ml (Fig. 3C to F). The addition of 100 ,ug of anti-CTB IgA to 1 ,ug of rhodamine-labelled CT had no direct effect on fluorescence intensity of the conjugate as monitored in an SLM 8000 fluorimeter (data not shown). Thus, the absence of fluorescence in monolayers treated with anti-CTB IgA was due to the lack of toxin binding to apical cell surfaces and not to nonspecific quenching of the rhodamine label by IgA binding. DISCUSSION We have generated 48 anti-CT hybridomas (27 IgA and 21 IgG) from Peyer's patch lymphocytes after peroral-systemic immunization with CT. All hybridoma cell lines produced antibodies directed against the CT B subunit, and the three monoclonal IgA antibodies studied in detail recognized at least two separate epitopes, neither of which corresponded to the GM1 binding site. When the three anti-CTB IgA antibodies were mixed with CT and applied to the apical surfaces of polarized, well-differentiated T84 intestinal cell monolayers, each was able to prevent the CT-induced Clsecretory response in a dose-dependent manner. By application of rhodamine-labelled CT to these monolayers, we showed that the IgA antibodies protected the cells by preventing adherence of toxin to apical plasma membranes. Immunization with CT resulted in the generation of CTspecific IgA and IgG committed lymphoblasts in the Peyer's patches. The fact that all anti-CT positive hybridomas that we obtained secreted antibodies directed against the B subunit and not the A subunit confirms previous oral immunization studies which showed the B subunit to be more antigenic in the mucosal immune system (13, 33). Indeed, even when rabbits were immunized subcutaneously with purified A-subunit preparations containing trace amounts of B subunit, the systemic immune response was directed toward the B subunit (33), and we have obtained IgG hybridomas against B but not the A subunit from spleen after systemic immunization with a commercial A-subunit preparation (18a). Together, these results confirm that the CT B subunit is a dominant immunogen in both systemic and mucosal immune systems. We have also confirmed the existence of antigenic differences between CT-1 and CT-2 produced by organisms of differing biotype and serotype: CT-1 is derived from a strain of classical biotype-Inaba serotype while the CT-2 producing strain is El Tor biotypeOgawa serotype (6). Previous checkerboard immunoblot analyses showed that mouse monoclonal IgG antibodies raised against these enterotoxins recognized various discon-

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FIG. 3. Monoclonal anti-CTB IgA inhibits the binding of rhodamine-labelled CT to the apical surfaces of T84 cell monolayers. Rhodamine-labelled CT was applied at a concentration of 1 ±g/fml to the apical surface of polarized, confluent T84 enterocytes for 1 h at 4°C in the presence of 100 ,ug of anti-CTB IgA or control anti-LPS IgA per ml. CT binding was assessed by examining 4-pm cryosections of the T84 cell monolayers by Nomarski optics (A, C, and E) and fluorescence microscopy (B, D, and F). Bar = 10 p.m. (A and B) Control anti-LPS IgA antibody did not block adherence of rhodamine-CT to the apical surfaces of T84 monolayers. (C and F) Anti-CTB monoclonal IgA antibodies completely blocked rhodamine-CT adherence (C and D, anti-CTB IgA-1; E and F, anti-CTB IgA-2).

tinuous epitopes, a subset of which were highly specific for the homologous antigen (17). We have now demonstrated that the IgA antibodies produced by mice in response to oral CT-1 also recognize unique as well as shared epitopes, a finding that is relevant to the choice of oral cholera vaccines for humans. The factors that protect intestinal epithelia in vivo against the effects of orally administered CT are not clearly defined. It has been suggested that non-immunoglobulin-mediated mechanisms may be important in protection against CTinduced fluid secretion even after toxin binding has occurred. One hypothesis is that the protection observed after CT immunization is due not to antibodies but to desensitization of adenylate cyclase (24), possibly mediated by a CT-inducible hormone-like antisecretory peptide (18). There is considerable evidence, however, that antitoxin antibodies play the primary role in protection against oral toxin challenge. Polyclonal anti-CTB antibodies have been shown to be capable of toxin neutralization in both rabbit skin permeability tests and ligated intestinal loop models (26, 33). Recently, it was demonstrated that protection of intestinal loops against intraluminally injected CT in animals orally immunized with CT is CD4 T cell dependent (14). There is a

direct correlation between the number of anti-CT secreting IgA plasma cells in the lamina propria and the inhibition of fluid secretion observed in the intestinal segment injected with toxin (27). Also, bile from animals immunized with CT protected intestinal segments from CT challenge, implicating anti-CT IgA as the protective factor (37). Our in vitro data are consistent with the in vivo studies cited above in which IgA was associated with protection against toxin challenge. The availability of well-differentiated T84 enterocytes grown in monolayer culture in the absence of other cells and tissues allowed us to directly measure the ability of anti-CTB IgA to inhibit CT action on intestinal enterocytes in the absence of other modulators. This in vitro system was recently shown to provide a very sensitive indicator of CT binding and action, since concentrations of toxin as low as 0.1 nM produced a reproducible change in Isc (19). All three of the monoclonal IgA antibodies analyzed here were able to prevent toxin binding to apical membranes of T84 epithelial cells and thus abrogate the CT-induced chloride secretory response. Neither of the two distinct epitopes recognized by the antibodies corresponded to the binding site for the cell surface receptor, GM1. Thus, it seems likely that binding of large, dimeric or polymeric IgA antibodies to the CT B

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subunits prevented toxin binding to cell surface GM1 by steric hindrance. The concentration necessary for protection in vitro was equivalent to four to five dimeric IgA antibodies per holotoxin molecule, a ratio that may reflect the pentameric nature of the B subunits of CT holotoxin (7). It is reasonable to assume that lower concentrations of IgA could protect the intestinal epithelium against CI in vivo, in the presence of clearance mechanisms such as mucus entrapment and peristalsis. It should be emphasized that demonstration of IgA protection against experimental challenge with CT does not establish the relative importance of anti-toxin IgA in protection against diarrheal disease caused by live V. cholerae organisms. Human volunteers orally immunized with purified CT and producing antitoxin antibodies were not protected against oral challenge with V. cholerae, whereas those immunized with vaccines containing vibrios were protected (20-23). In a companion study, we have shown that the monoclonal anti-CTB IgAs described in this report failed to protect suckling mice against a lethal oral dose of V. cholerae (1). Thus, the availability of hybridomas that secrete specific anti-CTlB IgA antibodies allows us to directly test and compare the capacity of anti-CTB to protect against secretory diarrhea in vitro and in vivo. ACKNOWLEDGMENTS

We are grateful to James Madara and Richard Weltzin for helpful discussions and to Charlene Delp for excellent technical assistance. This work was supported by National Institutes of Health research grants DK21505 and HD17557 (to M.R.N.), AI16716 and AI17312 (to R.A.F.), and AI18045 (to J.J.M.). Wayne Lencer is the recipient of NIH Clinical Investigator Award DK01848. Additional support was provided by NIH grant DK34854 to the Harvard Digestive Diseases Center. REFERENCES 1. Apter, F. M., P. Michetti, L. S. Winner Ill, J. A. Mack, J. J. Mekalanos, and M. R Neutra. 1993. Analysis of the roles of antilipopolysaccharide and anti-cholera toxin immunoglobulin A (IgA) antibodies in protection against Vibrio cholerae and cholera toxin by use of monoclonal IgA antibodies in vivo. Infect. Immun. 61:5279-5285. 2. Cash, R. A., S. I. Music, J. P. Libonati, J. P. Craig, N. F. Pierce, and R. B. Hornick. 1974. Response of man to infection with Vibrio cholerae. II. Protection from illness afforded by previous disease and vaccine. J. Infect. Dis. 130:325-333. 3. Cuatrecasas, P. 1973. Gangliosides and membrane receptors for cholera toxin. Biochemistry 12:3558-3566. 4. Dharmsathaphorn, K., and J. L. Madara. 1990. Established intestinal cell lines as model systems for electrolyte transport studies. Methods Enzymol. 192:354-359. 5. Donowitz, M., and M. J. Welsh. 1987. Regulation of mammalian small intestinal electrolyte secretion, p. 1351-1388. In L. R. Johnson (ed.), Physiology of the gastrointestinal tract. Raven Press, New York. 6. Finkelstein, R. A., M. F. Burks, A. Zupan, W. S. Dallas, C. 0. Jacob, and D. S. Ludwig. 1987. Epitopes of the cholera family of enterotoxins. Rev. Infect. Dis. 9:544-561. 7. Fishman, P. H. 1982. Role of membrane gangliosides in the binding and action of bacterial toxins. J. Membr. Biol. 69:85-97. 8. Gill, M., and R. Meren. 1978. ADP-ribosylation of membrane proteins catalysed by cholera toxin: basis of the activation of adenylate cyclase. Proc. Natl. Acad. Sci. USA 75:3050-3054. 9. Hansson, H. A., S. Lange, and I. LoAnnroth. 1984. Internalization in vivo of cholera toxin in the small intestinal epithelium of the rat. Acta Pathol. Microbiol. Scand. 92:15-21. 10. Herrington, D. A., R. H. Hall, G. A. Losonsky, J. J. Mekalanos, R. K. Taylor, and M. M. Levine. 1988. Toxin, toxin co-regulated pili and the toxR regulon are essential for Vibrio cholerae

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