Specific antibodies induced by nasally administered 40-kDa outer membrane protein of Porphyromonas gingivalis inhibits coaggregation activity of P. gingivalis

Vaccine 22 (2003) 250–256

Specific antibodies induced by nasally administered 40-kDa outer membrane protein of Porphyromonas gingivalis inhibits coaggregation activity of P. gingivalis Jun Namikoshi a , Shigeo Otake b,d , Satomi Maeba a , Mitsuo Hayakawa c,d , Yoshimitsu Abiko c,d , Masafumi Yamamoto b,d,∗ a

Clinical Pathology, Nihon University Graduate School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan Department of Oral Medicine, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan c Department of Biochemistry, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan b

d

Received 28 March 2003; received in revised form 24 June 2003; accepted 16 July 2003

Abstract In this study, we have assessed the efficacy of the 40-kDa outer membrane protein (40k-OMP) of Porphyromonas gingivalis as a nasal vaccine for the prevention of adult periodontitis. Mice nasally immunized with 40k-OMP and cholera toxin as mucosal adjuvant displayed significant levels of 40k-OMP-specific serum IgG1, IgG2b and IgA as well as mucosal IgA antibodies (Abs) in saliva and nasal secretions. Ab-forming cell (AFC) analysis confirmed the antibody titers by detecting high numbers of 40k-OMP-specific AFCs in spleen, salivary glands and nasal passages. Because 40k-OMP-specific IgG inhibited coaggregation of P. gingivalis vesicles and S. gordonii, it may be an important tool for the prevention of adult periodontitis. © 2003 Elsevier Ltd. All rights reserved. Keywords: Porphyromonas gingivalis; 40-kDa outer membrane protein; Mucosal vaccine

1. Introduction It is well established that Porphyromonas gingivalis, a gram-negative anaerobe, is one of the major pathogens associated with adult periodontitis, a disease which causes the destruction of the alveolar bone and, as a consequence, tooth loss [1]. Further, recent studies have suggested an association between periodontitis and systemic diseases including cardiovascular disease [2–6]. There are several mechanisms by which P. gingivalis causes disease [1], but perhaps the most important is coaggregation with other gram-positive bacteria. This coaggregation contributes to the formation and maturation of biofilm, which is known to cause both periodontal disease and dental caries [7–11]. In this regard, coaggregation between P. gingivalis and Actinomyces viscosus was found to be important to the initial events in the formation of subgingival biofilm [12–14]. Further, P. gingivalis adheres to S. gordonii, an early colonizer of tooth surfaces [8,9,11].

∗

Corresponding author. Tel.: +81-47-360-9432; fax: +81-47-360-9601. E-mail address: [email protected] (M. Yamamoto).

0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00576-0

The aggregation of P. gingivalis with and its attachment to other gram-positive or gram-negative bacteria are mediated by the specific outer membrane protein (OMP) in the whole cell-associated outer membrane or in the outer membrane vesicles [13]. The OMP produced by P. gingivalis resides both on the cell surface and in extracellular vesicles and has a molecular mass of 40-kDa outer membrane protein (40k-OMP) [15]. The 40k-OMP is a key virulence factor for coaggregation and is found on many strains of P. gingivalis [16–18]. Previous studies have shown that monoclonal antibodies (Abs) to recombinant (r) 40k-OMP are able to inhibit aggregation of several strains of P. gingivalis with A. viscosus [16,18]. Further, these Abs possess a complement-mediated bactericidal activity to P. gingivalis [19,20]. It has also been demonstrated that an anti-40k-OMP Ab opsonizes P. gingivalis as a target for phagocytosis by the human neutrophil cell line [21]. Finally, in an interesting study, human monoclonal Abs against r40k-OMP were generated by injecting severe combined immunodeficiency mice with human peripheral blood lymphocytes. These human monoclonal Abs against 40k-OMP proved to inhibit coaggregation of P. gingivalis vesicles with Actinomyces naeslundii [22]. Taken together, the above studies indicate

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that the induction of 40k-OMP-specific Abs to block or inhibit interbacterial adhesin is a logical approach for the prevention of colonization by P. gingivalis. Nasal vaccination, like other mucosal routes, offers several advantages when compared with parental immunization. First, nasal vaccination is capable of inducing both mucosal and systemic immune responses, which result in two layers of host protection against infectious diseases [23]. Second, this form of immunization offers advantages from a practical point of view. For example, needles and syringes are not required for nasal administration. These are extremely beneficial especially in developing countries. Thus, the major aim of the present study was to assess the potential of a nasal vaccine containing 40k-OMP to induce an immune response that would inhibit coaggregation activity of P. gingivlais.

coated with 40k-OMP (5 ␮g/ml) and blocked with 1% bovine serum albumin (BSA) and analyses were performed in duplicate. After blocking, serial dilutions of serum or saliva samples were added in duplicate. Starting dilutions of serum and saliva samples were 1:25 and 1:22 , respectively. Following incubation, the plates were washed and peroxidase-labeled goat anti-mouse ␮, ␥ or ␣ heavy chain-specific Abs (Southern Biotechnology Associates (SBA), Birmingham, AL) were added to appropriate wells. Finally, 2,2 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) with H2 O2 (Moss Inc., Pasadena, MA) was added for color development. Endpoint titers were expressed as the reciprocal log2 of the last dilution which gave an optical density at 414 nm of 0.1 greater than background after 15 min of incubation.

2. Materials and methods

2.5. ELISPOT employed for assessment of Ab-forming cells (AFCs)

2.1. Mice BALB/c mice were purchased from Sankyo Laboratories and were maintained in the experimental facility under pathogen-free conditions in the Nihon University School of Dentistry at Matsudo. All mice were provided with sterile food and water ad libitum and were used in this study at 8–12 weeks of age. 2.2. Antigen (Ag) and adjuvant The 40k-OMP was purified to homogeneity from a cell suspension prepared by sonication of Escherichia coli K-12 harboring the recombinant plasmid pMD125, as described previously [24]. The purity of the 40k-OMP was determined by SDS–PAGE, and no contaminating protein bands were noted. Further, possible residual endotoxin was assessed in the preparation with an LAL pyrochrome kit (Associates of Cape Cod Inc., Woods Hole, MA). The 40k-OMP contained as little as 0.4 pg of endotoxin. Cholera toxin (CT) was obtained from List Biologic Laboratories (Campbell, CA).

Single cell suspensions were obtained from salivary glands and nasal passages of immunized mice as previously described [28,29]. The mononuclear cells were obtained at the interface of the 40 and 75% layers of a discontinuous percoll gradient (Amersham Pharmacia Biotech, Piscataway, NJ). To assess numbers of Ag-specific AFCs, an ELISPOT assay was performed as previously described [30]. Briefly, 96-well nitrocellulose plates (Millititer HA; Millipore Corp., Bedford, MA) were coated with 40k-OMP (5 ␮g/ml), incubated for 20 h at 4 ◦ C, and the plates were then washed extensively and blocked with 10% goat serum. The blocking solution was discarded, and lymphoid cell suspensions at various dilutions were added to wells and were incubated for 4 h at 37 ◦ C in 5% CO2 in moist air. The detection Abs consisted of goat horseradish peroxidase-conjugated anti-mouse ␣ or ␥ heavy chain-specific Abs (SBA). Following overnight incubation, plates were washed with PBS and developed by addition of 3-amino-9-ethylcarbazole dissolved in 0.1 M sodium acetate buffer containing H2 O2 (Moss Inc.) to each well. Plates were incubated at room temperature for 15–20 min and washed with water, and AFCs were counted with the aid of a stereomicroscope.

2.3. Immunization and sample collection

2.6. Coaggregation assay

Mice were immunized nasally on days 0, 7 and 14 with a 10 ␮l aliquot (5 ␮l per nostril) of phosphate-buffered saline (PBS) containing 10 ␮g of 40k-OMP alone or combined with 1 ␮g of CT. Serum, saliva and nasal wash samples were collected, as described elsewhere [25], to examine 40k-OMP-specific antibody responses.

Serum IgG antibodies from immunized mice were purified using HiTrapTM protein G HP column (Amersham Biosciences, Piscataway, NJ). After purification, the protein levels of IgG were determined by BSA protein assay (Pierce Biotechnology Inc., Rockford, IL). P. gingivalis 381 and S. gordonii Challis were grown in brain–heart infusion (BBL Microbiology Systems, Cockeysville, MD) containing yeast extract (0.25%), haemin (10 ␮g/ml) and Vitamin K (1 ␮g/ml). The bacterial cells were incubated at 37 ◦ C in an anaerobic chamber containing N2 (80%), H2 (10%) and CO2 (10%). Vesicles were isolated from P. gingivalis as

2.4. Detection of Ag-specific Ab isotype responses The Ab titers in serum, saliva and nasal washes were determined by an ELISA [26,27]. Briefly, plates were

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described previously [13] with minor modifications. Briefly, P. gingivalis 381 cells from a 10 litre diffusate culture were removed from the growth medium by centrifugation. The supernatant containing vesicles was concentrated to 250 ml by passage through an ultrafiltration system (Millipore Co.). The sample was then dialyzed against 50 mM Tris–HCl containing 0.5 mM dithiothreitol at 4 ◦ C overnight to solubilize the pilli. The vesicles were collected by centrifugation and suspended in PBS. Coaggregation was determined by the visual assay method as described previously [12,16]. Briefly, P. gingivalis vesicles were pre-incubated with purified IgG Abs at 37 ◦ C for 30 min. The vesicle suspension (100 ␮l) was then mixed with equal volumes of S. gordonii suspension (approximately 1010 cells/ml) on the flocculation slide. The mixture was incubated at 37 ◦ C for 10 min with rotation. The degree of coaggregation was assigned a score from zero to four as previously described [31]. No visible coaggregation was scored as zero, and small uniform coaggregates as one. Scores of two or more were determined largely by the rate of settling of the coaggregates. Coaggregates which were easily seen but which did not settle immediately, leaving the suspension turbid, were scored as two. Large coaggregates which settled rapidly leaving supernatant fluid turbid were scored as three, and large coaggregates which settled immediately and left a clear supernatant fluid were scored as four. As a positive control, a monoclonal IgG Ab (Pg-ompA4) which was induced by immunization of female BALB/c mice with r40k-OMP as described previously [32] was applied. Briefly, cloned hybridoma cells were cultured with serum-free medium (Kyokuto Seiyaku, Tokyo, Japan). The monoclonal Ab in the culture supernatant was purified by passage through a column containing diethyl-aminoethyl cellulose (DE 52, Whatmann Ltd., Maidstone, Kent, UK), then dialyzed in PBS and concentrated in a microconcentrator (Amicon, Beverly, MA).

3. Results 3.1. Induction of 40k-OMP-specific antibody responses by nasal immunization with 40k-OMP To evaluate the ability of nasally administered 40k-OMP to induce serum Ab responses, a group of mice was nasally immunized with 40k-OMP alone. When the Ab-specific immune responses were analyzed by ELISA, IgM Ab responses were detected on day 21 (log2 , 8.1 ± 1.2), but serum IgG and mucosal IgA Abs were not (
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50

Number of AFCs/106 Cells

Reciprocal log2Titer

17

ND ND

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40 30 20 10 0

21

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Fig. 1. 40k-OMP-specific serum Ab isotype responses and numbers of IgG and IgA AFC in spleen. Groups of BALB/c mice were nasally immunized with 10 ␮g of 40k-OMP plus 1 ␮g of CT on days 0, 7 and 14. Serum samples were collected at weekly intervals and were assessed for 40k-OMP-specific IgM (open square), IgG (dotted square) and IgA (closed square) Abs (A). Mononuclear cells were isolated from spleen of mice on day 21 and examined for 40k-OMP-specific IgG (dotted square) and IgA (closed square) AFCs (B). The results are expressed as the mean ± S.E. obtained for six mice per group. ND: not detectable.

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Reciprocal log2Titer

17

14

11

8

5

IgG1 IgG2a IgG2b IgG3 Fig. 2. 40k-OMP-specific IgG subclass responses. Groups of BALB/c mice were nasally immunized with 40k-OMP plus CT as described in the legend of Fig. 1. IgG subclass responses were assessed on day 21. The results are expressed as the mean ± S.E. obtained for six mice per group.

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ing the coaggregation activity of P. gingivalis. To this end, P. gingivalis vesicles were pretreated with IgG from mice given 40k-OMP plus CT, 40k-OMP alone or PBS as control and then incubated with S. gordonii. The flocculation slide assay showed that the coaggregation activity of P. gingivalis vesicles with S. gordonii cells was inhibited by IgG Abs from mice given 40k-OMP plus CT in a dose-dependent fashion (Fig. 4A and B). Maximum inhibitory effect was obtained by 80 ng/␮l of IgG Ab from mice immunized with nasal 40k-OMP plus CT that was only two times higher when compared with the concentration of 40k-OMP-specific monoclonal IgG Ab (Fig. 4B). In contrast, IgG from mice given 40k-OMP alone only slightly inhibited the coaggregation activity of P. gingivalis. As expected, IgG from mice given PBS failed to suppress the coaggregation of P. gingivalis vesicles and S. gordonii (Fig. 4A and B).

4. Discussion

3.2. Nasally induced 40k-OMP-specific IgG suppresses coaggregation of P. gingivalis and S. gordonii In the next study, we determined whether Abs induced by nasally administered 40k-OMP were capable of suppress-

Reciprocal log2 Titer

8

6

4

2

(A)

ND

Nasal wash

ND

Previous studies have shown that 40k-OMP-specific rabbit monoclonal Abs inhibit the coaggregation activity of P. gingivalis [16,18,22]. Further, these Abs are known to possess complement-mediated bactericidal activity to P. gingivalis [19,20] and opsonic activity with respect to the human neutrophil function for the phagocytosis of P. gingivalis [21]. These findings have led to 40k-OMP being considered as a candidate Ab for the development of human vaccine. Our present study further explore its potential for human vaccines by using it as a model system to study the nature and kinetics of Ab-specific Ab responses induced by nasal immunization and their protective capability against oral infection by P. gingivalis. Our results demonstrate that nasal immunization with 40k-OMP vaccine induces significant 40k-OMP-specific IgM, IgG and IgA Ab responses in serum and IgA Abs in saliva and nasal secretions.

Number of AFCs/106 Cells

IgA Ab responses in saliva and nasal wash samples obtained a week after the third immunization (Fig. 3A), while no such responses (
800

600

400

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Saliva (B)

Nasal passage

Salivary gland

Fig. 3. Detection of 40k-OMP-specific IgA Ab responses in nasal washes and saliva (A) and numbers of IgA AFCs in nasal passages and salivary glands (B). Groups of BALB/c mice were nasally immunized with 40k-OMP plus CT (closed square) or 40k-OMP alone (open square) on days 0, 7 and 14. Saliva samples were collected at weekly intervals and were assessed for 40k-OMP-specific IgA Ab titers at day 21. Mononuclear cells from salivary glands were assessed for IgA AFCs. The results are expressed as the mean ± S.E. obtained for six mice per group. ND: not detectable.

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Fig. 4. Inhibition of coaggregation of P. gingivalis vesicles with S. gordonii by 40k-OMP-specific IgG Abs. P. gingivalis vesicles were pre-incubated with several concentrations of 40k-OMP-specific IgG and then mixed with S. gordonii on the flocculation slide. (A) The results of flocculation slide assay with (1) S. gordonii cells alone; (2) a mixture of S. gordonii cells and P. gingivalis vesicles; (3) a mixture of S. gordonii cells and P. gingivalis vesicles with 100 ␮g/ml of IgG Ab derived from serum of mice administered 40k-OMP alone; and (4) a mixture of S. gordonii cells and P. gingivalis vesicles with 100 ␮g/ml of IgG Ab derived from serum of mice given 40k-OMP plus CT. (B) Coaggregation score of S. gordonii and P. gingivalis vesicles treated with different concentrations of IgG Abs from mice given 40k-OMP plus CT (open square), 40k-OMP alone (open triangle) or PBS (open circle). As a positive control, monoclonal IgG antibody (Pg-ompA4) were applied (solid circle). IgG Abs used in this experiment were purified from pooled serum of three mice per group. The results were expressed as mean ± S.E. of four different experiments.

Furthermore, serum IgG anti-40k-OMP Abs were shown to significantly inhibit the coaggregation activity of P. gingivalis. These results suggest that 40k-OMP is an effective Ab for the induction of protective immune responses against P. gingivalis infection. It is well established that the mucosal immune system exists alongside and separate from the systemic immune system [23]. Therefore, the induction of systemic immune responses by systemic immunization does not result in significant mucosal immunity. However, mucosal immunization can and often does result in protective immunity not only in external secretions but in systemic compartments as well [38]. In many ways, the oral cavity is an important and characteristic compartment of the mucosal immune system. However, it differs from other mucosal compartments because its local immune responses are both mucosal and systemic. Local immune responses emanating from the salivary gland are part of the mucosal immune system while those emanating from the crevicular fluid derived from tissue fluids in blood capillaries are part of the systemic immune system [39]. Thus, although the main isotype of the oral cavity is secretory IgA, it is clear that systemic-derived IgG in crevicular fluid is also biologically active within the oral cavity. Further, IgG would play the more important role in the reduction of P. gingivalis colonization, since P. gingivalis is known to colonize both subgingival and supragingival plaque. Thus, effective protection against P. gingivalis infection requires both mucosal and systemic Ab responses. The immunization protocol used in this study was designed to induce significant 40k-OMP-specific Ab responses in mucosal secretion and serum. In this study, we chose the nasal delivery route because of its successful track record in inducing both mucosal and systemic immune responses [25,33,34,36,40].

Our results showed that nasal immunization with 40k-OMP vaccine induced mucosal IgA in saliva and nasal secretions as well as serum IgG and IgA Ab responses. Further, 40k-OMP-specific IgG Ab was found to inhibit the coaggregation activity of P. gingivalis. These findings suggest that the nasal administration of 40k-OMP can be considered to be a practical and effective route of immunization for the induction of specific immunity against P. gingivalis infection. Our results showed that 40k-OMP-specific IgG Abs inhibited coaggregation of P. gingivalis vesicles with S. gordonii. In this regard, S. gordonii is known to be one of the first bacteria to colonize newly cleaned teeth and is assumed to support the colonization of other bacteria [8,9,11]. Further, it has been shown that P. gingivalis adheres readily to the cell surface of S. gordonii [41]. It can then be concluded that S. gordonii supports the colonization of P. gingivalis and thus may develop periodontitis. Therefore, a logical approach to the prevention of P. gingivalis colonization would be to block or inhibit the interbacterial adhesion between S. gordonii and P. gingivalis, and that approach would have to include a consideration of 40k-OMP, which has been shown to play a key role in the coaggregation of P. gingivalis with other gram-positive bacteria [16–18]. In support of this, it has been shown that monoclonal Abs to 40k-OMP inhibit coaggregation of several strains of P. gingivalis vesicles with A. viscosus [16,18]. These studies together with our results suggest that nasally administered 40k-OMP may be an attractive candidate for an eventual periodontal disease vaccine. Examination of IgG subclasses showed that nasal 40k-OMP plus CT induced mainly IgG1 and IgG2b Ab responses. In this regard, CT is known to selectively induce Ab-specific Th2-type responses which promote IgG1 as well as mucosal IgA responses following nasal administration

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with protein Ab [25,37,42]. These results suggest that nasal immunization with 40k-OMP plus CT leads to the generation of Ab-specific Th2-type responses accounting for the induction of 40k-OMP-specific IgG1 and mucosal IgA responses in systemic and mucosal sites, respectively. It is important to note that mucosal immunization with 40k-OMP alone failed to enhance anti-40k-OMP Ab responses. Previous studies have likewise reported that protein antigen given via the mucosal route without adjuvant is only a weak immunogen [25,26,30,34,36,37,40]. Thus, a mucosal adjuvant such as CT is required for induction of 40k-OMP-specific Ab responses in both mucosal and systemic compartments when 40k-OMP is given by a mucosal route such as the intranasal one. However, despite these beneficial attributes, CT is unsuitable for use in humans, because the enzymatic activity of CT-A subunit lead to the development of a watery diarrhea [43]. In this regard, we generated mutants of CT, which harbor single amino acid substitutions in the ADP-ribosyltransferase active center that render them nontoxic [25,37,44,45]. Studies are underway to elucidate the efficacy of a combined nasal vaccine, i.e. 40k-OMP with nontoxic mutant CT, for the prevention of oral infection by P. gingivalis. In summary, our results provide evidence that nasal immunization with a vaccine consisting of 40k-OMP and the mucosal adjuvant CT elicited 40k-OMP-specific IgA Ab responses in saliva and nasal secretions as well as IgG and IgA in serum. Furthermore, the 40k-OMP-specific IgG Ab generated by nasal immunization was shown to inhibit the coaggregation of P. gingivalis vesicles with S. gordonii. The combination of 40k-OMP and CT provides a very effective means of eliciting protective levels of Abs to P. gingivalis, and therefore should be considered as a candidate Ab for prevention of P. gingivalis infection.

Acknowledgements We wish to thank Dr. Yasuko Shibata for purification of IgG, Dr. Koichi Hiratsuka for the coaggregation assay. We also thank Dr. Jerry R. McGhee for his critical review, and Dr. Kimberly McGhee for editing the manuscript. This work was supported by Grant-in-Aid for Scientific Research (A1, 13307060) and (B, 14370681) from Japan Society for the Promotion of Science, as well as Grant-in-Aid for Encouragement of Young Scientist (B, 13771105) and a grant from the Ministry of Education, Culture, Sports, Science and Technology to promote 2001-Multidisciplinary Research Projects.

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