INFECTION AND IMMUNITY, JUlY 1992, P. 2791-2799 0019-9567/92/072791-09$02.00/0

Vol. 60, No. 7

Copyright C) 1992, American Society for Microbiology

Epitope Mapping of Hemagglutinating Adhesin HA-Ag2 of Bacteroides (Porphyromonas) gingivalis MANON DESLAURIERS

AND

CHRISTIAN MOUTON*

Groupe de Recherche en Ecologie Buccale, Ecole de Medecine Dentaire, Universite Laval, Que6bec, Canada GIK 7P4 Received 22 October 1991/Accepted 31 March 1992

Thirteen monoclonal antibodies (MAbs) directed against hemagglutinating adhesin HA-Ag2 of Bacteroides (Porphyromonas) gingivalis were produced by immunizing mice with the relevant immunoprecipitate from crossed immunoelectrophoresis (CIE). Crossed immuno-affinoelectrophoresis and hemagglutination experiments confirmed that our MAbs recognized a molecule able to bind erythrocytes and involved in the hemagglutination process. In immunoelectron microscopy, these MAbs labelled amorphous material as novel cell-bound appendages distinct from fimbriae. CIE experiments allowed differentiation of the MAbs according to reactivity with immunoprecipitates Ag2, Ag8a, and Ag8c, which define HA-Ag2. The epitopes recognized by nine MAbs were mapped on three main antigenic domains (I, II, and III) by competition experiments and further grouped according to chemical composition and distribution on CIE immunoprecipitates. Domain I, defined by two MAbs, comprises an epitope with protein and carbohydrate determinants and distributed on Ag2 only. Epitopes of domain IIA, defined by four MAbs, are distributed on Ag8a, Ag8c, and Ag2 and are essentially composed of protein determinants but also have carbohydrate determinants that enhance the binding of the MAbs but are not essential. Epitopes of domain IIB, defined by two MAbs, and of domain III, defined by a single MAb, have a composition similar to that of domain IIA epitopes but are distributed on Ag8a and Ag8c only. A competition enzyme-linked immunosorbent assay with serum from normal subjects and patients with periodontitis suggested that domain I is more immunogenic than domain II. Bacteroides (Porphyromonas) gingivalis is a suspected major etiologic agent in adult periodontitis. Colonization of the subgingival area by this pathogen is a prerequisite for infection. This process is mediated by attachment of the microorganism to host cells and to other cells of the preexisting microbial flora (26). Structures that may be involved in attachment include fimbriae and other fibrillar appendages or adhesins, capsules, extracellular vesicles, and lipopolysaccharides (LPSs) (15, 16). A characteristic of B. gingivalis is its ability to bind to erythrocytes of humans and several animal species (20). Although some early studies indicated that fimbriae were responsible for this activity, it is now believed that B. gingivalis fimbriae are not responsible for hemagglutination (12, 29). Indeed, Mouton and coworkers (18) identified an outer membrane (OM) protein that is distinct from fimbriae and has an affinity for human erythrocytes. This adhesin, termed HA-Ag2, most likely involved in the hemagglutination process, was also shown to be a common antigen of the species (1). It is present in B. gingivalis extracellular vesicles, a fact that is of particular interest because these are suspected of playing an important role in colonization, inducing coaggregation of bacteria (14), promoting attachment to hydroxyapatite (2), and having hemagglutinating activity (8, 13). It was also recently shown that HA-Ag2 is an immunodominant antigen in patients with chronic periodontitis as well as in individuals with a healthy periodontium (3, 7), indicating its involvement in the host-pathogen relationship. In previous studies (1, 17, 18), the immunochemical characterization of HA-Ag2 was initiated with rabbit antisera raised against the relevant immunoprecipitate excised from *

crossed immunoelectrophoresis (CIE) gels. Polyclonal antisera to HA-Ag2 contain a variety of antibodies directed against different epitopes and are not suitable for fine characterization of the molecule. We therefore prepared a panel of monoclonal antibodies (MAbs) to immunologically characterize HA-Ag2, to localize it on the bacterial cell surface by electron microscopy, and to establish an epitope map of the molecule.

MATERIALS AND METHODS Bacterial strain and extracts. B. gingivalis ATCC 33277 in Trypticase-yeast extract broth supplemented with hemin (10 ,ug/ml) and vitamin K1 (5 ,ug/ml) in an anaerobic atmosphere (80% N2, 10% H2, 10% C02) for 24 to 48 h. A glass bead-EDTA extract (GBE) of the surface antigens of B. gingivalis was prepared as already described (23). OMs of B. gingivalis were obtained as extracellular vesicles (5, 8). For dot blot experiments, OMs were heated at 100°C for 5 min in Tris-buffered saline (TBS) containing either 2% sodium dodecyl sulfate (SDS) alone or 2% SDS and 5% ,B-mercaptoethanol, were digested with proteinase K at an enzyme/protein ratio of 1:10 (wt/wt) (10), or were oxidized with meta-periodate (22). Fusion and MAb production. MAbs were obtained by use of spleen cells from mice immunized with HA-Ag2 immunoprecipitate excised from CIE gels as already described (4). Spleen cells were fused with SP2/Ag14 myeloma cells at a ratio of 1:4 by use of 50% polyethylene glycol 4000. Hybridomas were tested in an enzyme-linked immunosorbent assay (ELISA) for antibodies against B. gingivalis GBE (4). Cells from positive wells were cloned twice by limiting dilution in microtiter plates. The resulting monoclonal cell lines were used to produce ascites fluid in pristane-primed BALB/c mice or grown in medium containing a low serum concenwas grown

Corresponding author. 2791

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DESLAURIERS AND MOUTON

tration to obtain MAbs from the growth supernatant. The immunoglobulin (Ig) subclass of each MAb was identified by Ouchterlony diffusion of the culture supernatant against Ig subclass-specific antisera (Sigma Chemical Co.). Immunoglobulin G (IgG) MAbs were purified on a hydroxyapatite column eluted with a phosphate gradient (9). IgM MAbs were used as concentrated growth supernatant. Hemagglutination inhibition. Twenty-five microliters of a cell wash extract (29) of B. gingivalis (known to possess hemagglutinating activity) was gently mixed with an equal volume of serially diluted MAbs (as purified IgG) in 50 mM phosphate-buffered saline (PBS) (pH 7.2) and incubated at room temperature for 1 h in a 96-well round-bottom microtiter plate. After incubation, 50 pl of a 0.5% suspension of fresh sheep erythrocytes in 50 mM PBS (pH 7.2) was added to the hemagglutinating extract-MAb mixture. After gentle mixing, the plate was incubated for 1 h at room temperature and overnight at 4°C. Inhibition was measured as the quantity (nanograms) of MAb as specific Ig needed to totally inhibit the hemagglutinating activity of the bacterial extract. Sera. Serum was obtained from 30 periodontitis patients (aged 35 to 69 years) in whom at least two pockets could be probed to 27 mm. Serum from normal subjects was obtained from 14 dental students with a healthy peridontium and excellent oral hygiene (aged 20 to 33 years). Two rabbit antisera, WL-303, monospecific for B. gingivalis HA-Ag2 (1), and 1373, produced against B. gingivalis GBE, were used in the second-dimension gel for some CIE experiments. CIE and CIAE. CIE and crossed immuno-affinoelectrophoresis (CIAE) were performed as previously described (17, 23). For CIAE, erythrocyte membranes were obtained as ghosts from human blood (28). SDS-PAGE, immunoblotting, and dot blotting. SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting were performed as described earlier (4). Antigen for dot blotting was deposited with a Bio-Dot microfiltration apparatus (Bio-Rad Laboratories) in accordance with the manufacturer's instructions. Native OMs, OMs heated in the presence of SDS alone or SDS and 1-mercaptoethanol, proteinase K-digested OMs, and meta-periodate-oxidized OMs diluted in TBS were applied at a concentration of 2 pLg of protein per well on a nitrocellulose membrane. After deposition of the antigen, the membrane was removed from the apparatus and cut into strips, each containing a dot of each extract. For antigen detection, unoccupied sites on the membrane were blocked by incubation for 1 h in TBS containing 3% fish gelatin and 0.05% Tween 20. The membrane was incubated with the primary antibody diluted in the same buffer. Unbound antibody was removed by washing for 30 min in TBS containing 0.05% Tween 20. Bound antibody was detected with a goat anti-mouse IgG-IgM-biotin-streptavidin-alkaline phosphatase system (Jackson ImmunoResearch Laboratories). Epitope analysis by a competition ELISA. The epitope specificities of the MAbs were compared with a competition ELISA and the additivity index (Al) described by Friguet et al. (6). A preliminary titration was conducted to determine the optimal dilution of each MAb needed to saturate its respective epitope when 0.4 pg of B. gingivalis OM per well was bound. This titration resulted in optical densities at 414 nm of between 0.15 and 0.8. Immulon-1 plates (Dynatech) were coated for 75 min at 37°C with a 100-,ul volume per well of 0.4 ,g of OM proteins diluted in 0.02 M carbonate buffer (pH 9.6). Unoccupied sites were quenched overnight at 4°C by the addition of 150 pA of 3% bovine serum albumin (BSA) in carbonate buffer per well. Wells were washed with PBS

INFECT. IMMUN.

containing 0.05% Tween 20. Twenty-five microliters of each MAb at twice the optimal dilution was added (yielding the saturation concentration once mixed), and the mixture was incubated for 1 h at 37°C. Wells were washed again, peroxidase-conjugated goat anti-mouse IgG (Jackson) diluted 1:8,000 was added to each well, and the mixture was incubated for 30 min at 37°C. After a final wash, 100 pA of ABTS [2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)] solution (400 ,ug/ml in phosphate-citrate buffer [pH 4.6] containing 0.15% H202) was added to each well, and the reaction was stopped after 15 min at 25°C by the addition of 50 plA of 10% SDS. To quantify the experimental results of this assay, we calculated an AI for each pair of antibodies as follows: Al = {[2A1+2/(A1 + A2)] - 1} x 100, where A1, A2, andA1+2 are the absorbances obtained in the ELISA with the primary antibody alone, the secondary antibody alone, and the two antibodies together, respectively. If the two antibodies are directed against different epitopes (no competition), A1+2 should be equal to the sum of A1 and A2 and the Al should approach 100%. However, if the two antibodies are directed against the same epitope (competition),A1+2 should be equal to the mean value forA1 and A2 and the AI should tend to be 0%. Multiple replicates of each MAb pair were assayed, and a t test was used to determine which Al values did not differ significantly (a = 0.05) from 0 or 100%. Competition ELISA with human sera. To determine whether an epitope was recognized by human Ig, we measured the inhibition of MAb reactivity produced by adding serially diluted human sera. Optimal dilutions of reagents were determined as stated above. The assay used essentially the same protocol as the binding site assay; however, serial dilutions of human sera were mixed with each MAb at its saturation dilution. After 1 h of incubation at 37°C, goat anti-mouse IgG conjugated to peroxidase was added, and the mixture was incubated for 30 min at 37°C before substrate was added. For each serum, the dilution yielding a 50% reduction in MAb binding was calculated from plots of optical densities at 414 nm against serum dilutions. Electron microscopy. A bacterial suspension was deposited on Formvar-coated copper grids at room temperature. To block unoccupied sites, we incubated air-dried grids successively with avidin and biotin for 20 min each at 37°C, with three rinses in PBS and two rinses in distilled water for 1 min each after each incubation. The grids were incubated with MAbs diluted in PBS containing 0.5% BSA (PBS-BSA) for 30 min at 4'C. After the rinsing procedure, the grids were incubated with biotinylated goat anti-mouse Ig (Jackson) diluted in PBS-BSA for 30 min at 4°C and rinsed again. Before incubation with colloidal gold probe, the grids were incubated with PBS containing 0.1% polyethylene glycol 3350 for 10 min. Streptavidin conjugated to 5-nm colloidal gold particles (EY Labs, San Mateo, Calif.) was diluted in PBS containing 1% gelatin and 1% BSA, and the mixture was incubated for 30 min at 4°C. After a final rinse procedure, samples were negatively stained with 2% phosphotungstic acid. RESULTS Characteristics of the MAbs. We obtained 13 MAbs specific for HA-Ag2 from three different fusions with splenocytes from mice immunized with the relevant immunoprecipitate from CIE (Table 1). Eleven of these MAbs were of the IgGl subclass, and two were of the IgM class. Nine of the MAbs totally inhibited the hemagglutination of fresh

VOL. 60, 1992

EPITOPES OF B. GINGIVALIS HEMAGGLUTININ

TABLE 1. Properties of MAbs obtained by injection of a CIE immunoprecipitate MAb

11C4 llFl

15B2 12D2

11B9 16C6 18F6

lSG1 23H5 31A10 31C10 33H5 35D7

Isotype

IgGl IgGl IgGl

IgGI IgGl IgGl IgM IgGl IgGl IgGl IgM IgGl

IgM

ng of Ig needed for hemagglutination inhibitiona

48 25 63 13 27 25 NDe 10 15 ND ND 12 ND

Result in:

CIEb Ag8

Ag2

+ + + + + + + +

+

+ +

-

+

+ + + + + + + +

CIAEc

EMd

+ + + + + + ND +

+ + + + + + + + ND ND ND ND ND

NPf ND ND NP ND

a Nanograms of Ig needed to totally inhibit the hemagglutinating activity of a B. gingivalis extract. b +, modifications of the migration of CIE immunoprecipitate(s) Ag8 and/or Ag2 (see Results for details); -, no modifications. c +, recognition of an antigen whose anodal migration is retarded in CIAE with erythrocyte membranes. d +, labelling of B. gingivalis appendages in electron microscopy. e ND, not done. f NP, nonprecipitating antibody.

sheep erythrocytes induced by B. gingivalis surface components at concentrations ranging from 10 to 63 ng of IgG. A control consisting of an irrelevant mouse MAb (noncommercial IgG3; anti-mouse NK 4 cells) proved that this inhibition was not an artifact due to the interfering presence of mouse antibodies. To assess the specificities of the MAbs for the immunizing antigen, we verified their capacity to lower the position in gels of the immunoprecipitate corresponding to HA-Ag2 in CIE with an intermediate gel. A set of typical results is shown in Fig. 1. MAbs 23H5 (Fig. 1C) and 33H5 and 35D7 (data not shown) retarded the migration of Ag2 but had no other effect. Surprisingly, MAbs 15B2, 11C4, 15G1, and 16C6 not only lowered the position of the Ag2 immunoprecipitate but also caused the dissociation of another immunoprecipitate, Ag8. It was thus revealed that Ag8 consists of at least three distinct components, Ag8a, Ag8b, and Ag8c, which comigrate as a single immunoprecipitate upon reaction with polyclonal antiserum. When the MAbs were added to the intermediate gel, Ag8b retained the same position, but the migration of Ag8a and Ag8c was retarded. A typical example of the modification of the migration pattern is shown in Fig. 1D with MAb 11C4. Mabs 12D2, llFl, and 11B9 allowed the resolution of Ag8 into three components but did not retard the migration of Ag2 (data not shown). The modifications of the CIE migration patterns are reported in Table 1. To confirm that the MAbs recognized an antigen able to bind erythrocyte membranes, we used CIAE. The seven MAbs tested precipitated an antigen whose migration was retarded by erythrocyte membranes. A set of typical results is shown in Fig. 2 with MAb 15B2, and all CIAE results are listed in Table 1. Surprisingly, only MAb 35D7 reacted appreciably with electrophoretically separated OMs in immunoblots, recognizing two bands at 43 and 49 kDa and an additional band at 68 kDa (Fig. 3). The other MAbs showed no reaction or faint

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reactions, suggesting that degradation of the target epitopes occurred upon SDS-PAGE sample preparation. Characteristics of the epitopes recognized by the MAbs. To investigate the nature of the epitopes recognized by the MAbs, we performed dot blot experiments with B. gingivalis OMs under different conditions: native, heated at 100°C in SDS with and without ,-mercaptoethanol, digested with proteinase K, and oxidized with meta-periodate. These experiments indicated that the epitope recognized by MAb 35D7 and that recognized by the control MAb, 8C2, which is directed to B. gingivalis LPS, were the only ones not destroyed by heat, as shown in Fig. 4. All reactivities were also greatly diminished by proteinase K treatment, with the same two exceptions. Reactivities with all MAbs were reduced by treatment with meta-periodate, and the reduction was especially pronounced for three MAbs (23H5, 33H5, and 35D7) and for the anti-LPS MAb. These results suggest that our MAbs are directed to a proteinaceous antigen that contains carbohydrate residues. These residues would be essential for the binding of MAbs 23H5, 33H5, and 35D7 but not the other MAbs. Epitope specificities of the MAbs. To determine whether the MAbs bound to the same or unrelated epitopes, we used a competition ELISA and an Al. A saturating amount of each MAb was added in combination with each of the other MAbs to B. gingivalis OM-coated wells, and the total amount of bound MAb was determined by the ELISA. In this procedure, only MAbs of the IgG isotype could be used, because steric hindrance induced by the IgM molecule makes it unsuitable for the competition assay. MAb 31A10 was not used because of its low affinity. The mean AI values (two to nine replicate assays for each pair) obtained with each of the 36 MAb pairs tested are listed in Table 2. We established (t test, ao = 0.05) that little or no inhibition of one MAb by another with an Al of .79% was not significantly different from complete inhibition (Al = 100%) and meant the recognition of separate and topographically distinct epitopes. Inhibition of binding with an Al of