INTRODUCTION

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It is now well recognized that the adherence of bacteria to host tissues is a prerequisite for colonization and one of the causative factors of bacterial pathogenesis. The bacterial colonization of gingival tissues is critical in the pathogenic process of periodontal disease resulting in tissue destruction. Porphyromonas gingivalis has been implicated as a pathogen in the development of adult periodontitis, a chronic inflammatory disease of the supporting tissues of the teeth that leads to tooth loss (8, 12, 32, 33). Oral infection of nonhuman primates by P. gingivalis caused destructive disease in a ligature-induced model of periodontitis (7, 8, 22). However, the mechanisms by which P. gingivalis colonizes tooth surfaces and the adjacent periodontal tissues remain largely uncharacterized. Recently, various molecules present at the surface of this bacterium, such as fimbriae and vesicles, and potential molecular adhesins, including lectins, hemagglutinins, and lipopolysaccharide, have been characterized for their roles of adhesion (2, 9, 12, 15). Among these, the hemagglutinin is the major glycoprotein of bacterial vesicles (27) and may mediate the adsorption and penetration of bacteria into host cells (14, 24). Some hemagglutinin domains are encoded by a portion of a protease gene and possess the ability to degrade a broad range of host proteins, including structural and defense proteins (4, 5, 18, 28, 30), while the multivalent hemagglutinin is encoded by a different gene, such as hagA, which is larger than the protease genes (10). The hemagglutinin gene and other protease genes may share the hemagglutinin domain sequence from a multigene family (10, 19, 31).

Several investigators have attempted both active and passive immunization of nonhuman species and human to protect against periodontal disease by using antibodies against P. gingivalis (1, 7, 11, 26, 29). However, because of the sizes of the molecules and their inability to penetrate into tissue, the use of intact antibodies in humans may have several unexpected disadvantages. New technology using the single-chain variable fragment (ScFv) has been developed to overcome these problems (13, 34). This method relies on the single amino acid chain being expressed from the DNA in which two cDNAs specifying the variable (V) regions of both heavy (H) and light (L) chains are connected in frame to a linker sequence that encodes a flexible peptide.

To establish the passive immunization system in humans, we focused on the construction of ScFv antibody against the P. gingivalis hemagglutinin. We isolated a mouse monoclonal antibody (MAb) and prepared the ScFv antibody. The recombinant ScFv antibody produced from Escherichia coli cells inhibited the P. gingivalis vesicle-associated hemagglutinating activity.

	MATERIALS AND METHODS

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Materials. The recombinant phage antibody system (mouse ScFv module, expression module, and detection module) and protein G-Sepharose 4 Fast Flow were purchased from Pharmacia Biotech (Uppsala, Sweden). The Taq DyeDeoxy terminator cycle sequencing kit for DNA sequencing and Taq DNA polymerase were from Applied Biosystems Inc. (Foster City, Calif.). The Geneclean II kit was from Bio 101 (Vista, Calif.). Other chemicals used in this study were of analytical grade.

Preparation of vesicles. P. gingivalis 381 was grown in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) supplemented with hemin (19 mg/ml) and vitamin K₁ (1 mg/ml) in an anaerobic atmosphere (80% N₂, 10% H₂, 10% CO₂) for 24 to 48 h. Vesicles were isolated by the method of Grenier and Mayrand (9), with slight modification. Briefly, P. gingivalis 381 cells from a 10-liter diffuse culture (3-day culture) were removed from the growth medium by centrifugation (10,000 × g for 30 min). The supernatant containing the vesicles was concentrated to 250 ml by passage through an ultrafiltration system (Millipore Co., Bedford, Mass.) with a membrane having a molecular weight cutoff of 10,000. This sample was dialyzed against 50 mM Tris-HCl (pH 9.5) containing 0.5 mM dithiothreitol at 4°C overnight to solubilize the pili. The vesicles were collected by centrifugation (90,000 × g) for 2 h and suspended in Dulbecco's phosphate-buffered saline solution without Mg²⁺ and Ca²⁺ [PBS()]. This suspension was dialyzed against PBS() at 4°C overnight and kept at 20°C until used. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), P. gingivalis vesicles were solubilized in 0.1% SDS solution.

Generation of hybridoma antibody against the vesicles of P. gingivalis. Six- to eight-week-old BALB/c mice were injected with 200 µg of vesicles in Freund's complete adjuvant. Three times injections were given with the same amount of vesicles. First and second immunization were peritoneal cavity administration at 14-day interval. After another 14 days, a third injection was given through a tail vein. Four days later, the spleen cells of immunized mice were fused with SP2/Ag14 myeloma cells (1:5) in 50% polyethylene glycol 4000 (Sigma Chemical Co., St. Louis, Mo.). The hybridomas were tested by enzyme-linked immunosorbent assay (ELISA) for production of antibodies against solubilized P. gingivalis vesicles. Cells from positive wells were cloned twice by limiting dilution in microtiter plates. The resulting monoclonal cell lines were grown in medium containing a low serum concentration to obtain MAbs from the growth supernatant.

Construction of an ScFv antibody. A schema representing the procedure used for cDNA cloning and construction of recombinant ScFv antibody is shown in Fig. 1. Total RNAs from hybridoma cells were isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction method (3). Twenty micrograms of total RNA was used as a template for the reverse transcriptase reaction. First-strand cDNAs were synthesized by using primed first-strand reaction mixtures. The cDNAs coding for V regions of the H and L chains (V_H and V_L, respectively) were then amplified by PCR using a set of primers which were included in the mouse ScFv module/recombinant phage antibody system (Pharmacia Biotech). PCR amplification was run for 30 cycles (94°C for 1 min; 55°C for 2 min; 72°C for 2 min). Amplified DNAs of V_H and V_L fragments were purified separately by agarose gel electrophoresis to remove primers from amplification products.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Overview of the production of recombinant ScFv antibody. For details, see text.

Preparation of anti-E tag antibody affinity column. Protein G-Sepharose 4 Fast Flow was equilibrated with 20 mM sodium phosphate buffer (pH 8.2). Diluted anti-E-tag antibody (5 mg) with 5 ml of 20 mM sodium phosphate buffer (pH 8.2) was added to the gel matrix in the column. This mixture was incubated for 2 h at room temperature with continuous gentle mixing by inversion. Next, the gel matrix was washed with 20 mM sodium phosphate buffer (pH 8.2), treated with 40 mg of dimethyl pimelimidate dihydrochloride dissolved in 10 ml of 0.2 M triethanolamine-HCl (pH 8.2), and allowed to stand for 15 min. The column was completely washed with 0.1 M glycine-HCl (pH 2.8) to remove any anti-E-tag antibody that was not covalently bound. Finally, 20 mM phosphate buffer (pH 8.2) was used to neutralize the gel matrix. This column was equilibrated with 10 bed volumes of PBS() when ScFv antibody fraction was purified.

Inhibition of hemagglutinating activity. Ten microliters of vesicle solution (250 ng of protein/ml) and ScFv antibody solution (40 µl) were transferred to microtiter wells and incubated at a room temperature for 30 min with gentle shaking. Then 150 µl of human erythrocytes (2.7 × 10⁵ cells) was added, and inhibition of hemagglutinating activity was assessed by photography following 1 h of incubation at room temperature.

Sequence analysis of ScFv H and L chains of pMDABG2-4. Plasmid pMDABG 2-4 was extracted, and nucleotide sequencing was carried out by double-stranded dideoxynucleotide sequencing in both directions, using a Taq DyDeoxy terminator cycle sequencing kit (Applied Biosystems). The primers S1 (5'-CAA CGT GAA AAA ATT ATT ATT CGC-3'), S3 (5'-GGT TCA GGC GGA GGT GGC TCT GG-3'), S4 (5'-CCA GAG CCA CCT CCG CCT GAA CC-3'), and S6 (5'-GTA AAT GAA TTT TCT GTA TGA GG-3') were complementary to the vector and linker DNA sequences. The sequence data obtained were subjected to a homology search using the GenBank and IMGT (integrated database for immunogenetics; http://www.genetik.uni-koeln.de/dnaplot/) databases.

Nucleotide sequence accession number. The nucleotide sequence of ScFv-MDABG2-4 has been registered under DDBJ accession no. AB007986.

	RESULTS

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Isolation and characterization of a MAb against P. gingivalis vesicles. Several hybridoma clones which recognize the P. gingivalis vesicles by ELISA were constructed, and three of the clones were selected in two steps: (i) Western blot analysis using the P. gingivalis vesicles and (ii) assay of the hemagglutination inhibition activity of P. gingivalis vesicles. As shown in Fig. 2A, the one of the three MAbs produced against the vesicles of P. gingivalis, designated MAb Pg-vc, strongly recognized the 43- and 49-kDa bands of the vesicle fraction by Western blot analysis. Furthermore, this blotting profile was exactly the same as that of the anti-HA-Ag2 antibody (kindly supplied by C. Mouton [6]), which also strongly recognized both 43- and 49-kDa proteins corresponding to hemagglutinating adhesin.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Immunological (A) and biological (B) activities of MAb Pg-vc. (A) To assess the immunological activity of MAb Pg-vc against the proteins isolated from P. gingivalis, Western blot analysis was carried out. For comparison, anti-HA-Ag2 Ab was used. Lanes 1, cell lysate; 2, vesicle fraction. (B) To examine the biological activity of MAb Pg-vc, 10 µl of vesicle solution (250 ng of protein/ml) and MAb Pg-vc in wells (total volume, 40 µl) were incubated for 30 min at room temperature, and then 150 µl of erythrocyte suspension (2.7 × 105 cells) was added.

Preparation of the ScFv fragment. After first-strand synthesis using 20 µg of total RNA purified from the hybridoma cells, the cDNAs of V_H and V_L were amplified by PCR. As shown in Fig. 3, approximately 340-bp (V_H) and 320-bp (V_L) products were visualized on the electrophoresed agarose gel (lanes 1 and 2). The purified cDNAs of V_H and V_L were assembled into a single gene by using a DNA linker fragment, and the PCR-amplified ScFv DNA fragment (approximately 750 bp) was obtained (lane 3). To separate the expected assembled products, the 750-bp cDNA band was purified with a Geneclean II kit. Following digestion with the restriction enzymes SfiI and NotI, the ScFv DNA fragment was ligated to a phagemid vector, pCANTAB5E. After transformation of E. coli HB2151, a total of 192 E. coli transformants harboring chimeric plasmids with inserts of approximately 750 bp were isolated. Cell lysates were obtained from eight of these transformants, and immunological properties were further examined by Western blotting against the P. gingivalis vesicles. Finally, we obtained the E. coli transformant harboring plasmid pMDABG2-4, which expressed the recombinant ScFv antibody (ScFv-MDABG2-4) against the P. gingivalis vesicles.

View larger version (70K): [in this window] [in a new window]   FIG. 3.   Agarose gel electrophoresis of DNA fragments. Lanes: 1 and 2, PCR products of VH, and VL, respectively; 3, ligation mixture of VH and VL; 4, size marker (100-bp ladder DNA); 5, SfiI and NotI digest of pScFv-MDABG2-4. The arrow indicates the ScFv fragments.

Purification of the ScFv-MDABG2-4. To purify the recombinant ScFv protein, we first examined the location of this protein in the E. coli transformant. As shown in Fig. 4A, this protein existed in both the periplasmic space and cell lysate. Subsequently, large-scale production of ScFv-MDABG2-4 was performed with a periplasmic extract fraction. The periplasmic fraction from 1 liter of cell culture was purified by the anti-E-tag antibody affinity column. This protein (31 kDa) was purified by passage through the anti-E-tag antibody affinity column (Fig. 4B).

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Location and purification of the ScFv-MDABG2-4. (A) To identify the location of the recombinant ScFv-MDABG2-4 protein, culture supernatant (lane 1), periplasmic fraction (lane 2), and cell lysate (lane 3) were prepared from an E. coli transformant, and Western blot analysis was carried out. Five milliliters of the overnight culture of clone MDABG2-4 was inoculated into 50 ml of fresh SB medium (35 g of tryptone, 20 g of yeast extract, and 5 g of NaCl in 1 liter of water) with 100 µg of ampicillin per ml and incubated for 1.5 h at 30°C. The expression of ScFv protein was induced by the addition of 1 mM IPTG and incubation for 3 h at 30°C. Supernatant was obtained by centrifugation of the culture medium. Cell pellets were resuspended in 0.5 ml of PBS()1 mM EDTA for 10 min on ice and then centrifuged to obtain the periplasmic fraction. Other pellets of the same cells were resuspended in 0.5 ml of PBS() and boiled for 5 min. This supernatant after centrifugation is showed in lane 3 (as the cell lysate). The ScFv protein produced was detected by using the anti-E-tag antibody, since the ScFv antibody was a protein fused with the E-tag peptide. (B) Affinity-purified ScFv-MDABG2-4 examined by Coomassie brilliant blue staining (CBB) or by Western blot analysis using an anti-E-tag antibody (Western-blot).

Characterization of ScFv-MDABG2-4. To assess the immunological activity of ScFv-MDABG2-4, Western blot analysis was carried out. For comparison, MAb Pg-vc, was also used. The solubilized vesicle proteins were transferred onto a nitrocellulose membrane and allowed to react with both antibodies. As shown in Fig. 5, MAb Pg-vc recognized several proteins, including 43- and 49-kDa proteins, and ScFv-MDABG2-4 could react with four protein blots, which were also recognized by parental MAb Pg-vc.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Immunological activity of ScFv-MDABG2-4. The P. gingivalis vesicle fraction (2 µg of protein/lane) was separated by SDS-PAGE (12% gel) and transferred onto a nitrocellulose membrane. After overnight blocking with 5% skim milk solution, the membrane was exposed with ScFv-MDABG2-4 antibody (1:50 dilution with 5% skim milk solution; the eluted fraction [fraction 5] of the anti-E-tag antibody affinity column in Fig. 4B was directly used as ScFv-MDABG2-4) for 16 h. Next, the membrane was incubated with the mouse anti-E-tag antibody and then a goat anti-mouse IgG antibody (horseradish peroxidase conjugated) for 2 h. Protein bands were visualized by the standard method using horseradish peroxidase. For comparison, another membrane was exposed with MAb Pg-vc for 16 h, and protein bands were detected in the same way.

View larger version (64K): [in this window] [in a new window]   FIG. 6.   Biological activity of ScFv-MDABG2-4. To evaluate the biological activity of ScFv-MDABG2-4, indicated volumes of affinity-purified protein were added to microtiter wells, and inhibition of hemagglutinating activity was assayed as described in the legend to Fig. 2B. The eluted fraction (fraction 5) of anti-E-tag antibody affinity column in Fig. 4B was directly used as ScFv-MDABG2-4.

Nucleotide sequence analysis. To identify the gene families for the V_L and V_H regions, nucleotide sequencing was carried out. As shown in Fig. 7, the deduced amino acid sequence of ScFv-MDABG2-4 confirmed the expected protein structure where V_L and V_H regions were connected with three consecutive GGGGS repeats which resulted from the linker sequence. Comparison of the nucleotide sequence with those deposited in the IMGT database revealed that the V_H and V_L regions were highly homologous to the murine VH1 and the mouse V12/13 families, respectively. Interestingly, homology search using the GenBank database indicated that ScFv-MDABG2-4 resembled the antigenic site of the A/PR/8/34 influenza virus hemagglutinin (data not shown; see reference 17).

View larger version (43K): [in this window] [in a new window]   FIG. 7.   DNA and deduced amino acid sequences of pMDABG2-4. The three boxes in the area of the H and L chains of pMDABG2-4 indicate the CDRs in the V region of the antibody.

	DISCUSSION

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Various P. gingivalis hemagglutinins and aggregating factors with different biochemical properties and molecular weights have been reported by several research groups. Recently, several investigators demonstrated that fimbiae were involved in the adhesion of P. gingivalis but not in the agglutination of erythrocytes (15, 35). Deslauriers and Mouton have purified the surface protein complex, hemagglutinating adhesin HA-Ag2 (6), from vesicles capable of binding erythrocytes and demonstrated that this protein complex was distinct from fimbriae (25). Moreover, they showed that HA-Ag2 was composed of two proteins (43 and 49 kDa), while the fimbrilin subunit had an apparent molecular mass of 42 kDa in all extracts tested. As expected, the MAb against fimbriae inhibited the adhesion of P. gingivalis to epithelial cells but was unable to inhibit hemagglutinating activity (15, 35). MAb Pg-vc and the recombinant ScFv protein, ScFv-MDABG2-4, derived from it possessed almost the same immunological and biological properties; these two antibodies recognized two proteins, of 43 and 49 kDa, in Western blot analysis as anti-HA-Ag2 antibody did and inhibited the hemagglutinating activity caused by the P. gingivalis vesicles in human erythrocytes (Fig. 2, 5, and 6).

Several groups have attempted to administer MAbs to protect against disease. In experimental immunization for dental caries, MAbs specific to Streptococcus mutans or bovine milk containing antibodies to S. mutans could prevent the colonization of microorganisms and the development of caries in nonhuman primates or gnotobiotic rats (23). Furthermore, using MAbs against P. gingivalis, Booth et al. succeeded in passive immunization to prevent colonization of P. gingivalis for up to 9 months (1). However, unexpected disadvantages may be apparent if intact antibodies of nonhuman origin are used. To overcome this problem, one potential approach is the construction of the chimeric MAbs in which murine-origin V_H and V_L regions are combined with the constant (C) regions from human sequences (16, 20). In this "humanized" MAb, only the antigen recognition sites (complementarity-determining regions [CDRs]), were of nonhuman origin, whereas all frameworks in V and C regions were products of human genes. On the other hand, Ma et al. have demonstrated the expression of chimeric antibody in a plant (21). Four transgenic Nicotiana tabacum plants that expressed murine kappa-chain, hybrid IgA-G heavy-chain, a murine joining-chain, and rabbit secretory component MAbs, respectively were generated. Following the cross-breeding of these four plants in vivo, these chains were assembled into a functional secretory immunoglobulin that recognized the native streptococcal antigen I/II, the adhesion molecule present at the surface of bacterial cells. The functional recombinant ScFv antibody against the P. gingivalis hemagglutinin shown in this present study may be useful for establishing a passive mucosal immunotherapy since it is relatively easy to prepare large quantities of purified protein.

We have determined the nucleotide sequences around both the V_H and V_L regions and elucidated the gene families of these regions. There are no sequence data that completely match the V region of this antibody in the databases. However, the V_H region exhibited a high degree of homology with that of the antigenic site of the influenza virus A/PR/8/34 hemagglutinin (17). The compilation of these data may help clarify the stereochemistry between antibody CDRs and antigen epitopes and thus be essential in designing novel antibody for establishing a passive immunization system.

In conclusion, we succeeded in expressing a functional ScFv antibody against the P. gingivalis hemagglutinin-like molecules, and this system could provide an abundant source of immunotherapeutic agent for protecting against periodontal diseases.