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J Dent Res 86(5):446-450, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Arg-gingipain A DNA Vaccine Prevents Alveolar Bone Loss in Mice

K. Miyachi1, K. Ishihara1,2,*, R. Kimizuka1, and K. Okuda1

1 Department of Microbiology and
2 Oral Health Science Center, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba, 261-8502, Japan

* corresponding author, ishihara{at}tdc.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One major pathogenic factor of Porphyromonas gingivalis is Arg-gingipain (Rgp), an arginine-specific cysteine proteinase. To clarify the effect of rgpA DNA vaccine, we immunized BALB/c mice via the abdomen with a Gene Gun or via the nasal cavity weekly for 6 weeks. After immunization, the mice were challenged orally with P. gingivalis. Immunization elicited IgG responses against P. gingivalis in both groups. Nasal immunization also induced sIgA against P. gingivalis, although Gene Gun immunization did not. Reduction of alveolar bone loss was observed in both groups at 42 days following initial infection. This effect was more pronounced in the intranasal immunization group than in the Gene Gun group. The results of this study suggest that immunization with rgpA DNA vaccine via the nasal cavity is an effective method for preventing alveolar bone loss incurred by infection with P. gingivalis.

KEY WORDS: Porphyromonas gingivalis • vaccine • periodontitis • bone loss • mucosal immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, has emerged as a leading candidate pathogen in the development of chronic periodontitis (Socransky et al., 1998). This microorganism possesses multiple pathogenic factors such as lipopolysaccharides, capsular polysaccharides, fimbriae, hemagglutinins, and proteases (Mayrand and Holt, 1988). Among these, gingipains, a group of cysteine proteases, are major weapons in its arsenal of attack on the periodontal region. Gingipains consist of Arg-gingipains (RgpA and RgpB) and Lys-gingipain (Curtis et al., 1999). They are believed to play a major role in the pathogenesis of periodontitis, degrading a variety of host proteins. In this way, they dysregulate the host defense mechanisms, resulting in tissue destruction and alveolar bone resorption (Grenier et al., 2003; Houle et al., 2003; Imamura, 2003; Goulet et al., 2004).

Immunization studies with P. gingivalis in animal models have produced encouraging results which suggest that vaccine-elevated IgG levels can protect against periodontal disease (Gibson and Genco, 2001; Rajapakse et al., 2002; Gonzalez et al., 2003). Mucosal immunity, in which sIgA is a key factor, is the first line of defense against pathogens, working to prevent systemic or local infection. We previously reported that immunization of mice with rgpA DNA vaccine elicited high levels of specific IgGs against P. gingivalis (Yonezawa et al., 2001). In this study, we evaluated the effect of intranasal (i.n.) immunization with a DNA vaccine.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Vaccination with Gene Gun and HVJ Envelope Vector
Approval to conduct this study was obtained from the Animal Use Committee of Tokyo Dental College. A six-week-old female BALB/c mouse (Sankyo Laboratories Service Co. Inc,, Tokyo, Japan) was used for each vaccination.

Immunization with the rgpA DNA vaccine was performed as described previously (Yonezawa et al., 2001). Briefly, pVax1 (Invitrogen, Carlsbad, CA, USA) harboring the whole rgpA open reading frame from P. gingivalis ATCC33277 was used as the rgpA DNA vaccine. Gold particles (1.6-µm) were coated with the vaccine according to the manufacturer’s instructions. A Helios Gene Gun (Bio-Rad Laboratories, Hercules, CA, USA) was used to give the mice a total of 2.5 µg DNA via the skin of the abdomen at wks 0, 1, 2, 3, 4, 5, 6, and 7.

Intranasal immunization with the rgpA DNA vaccine was performed with the hemagglutinating virus of Japan (HVJ) envelope vector kit (Ishihara-Sangyo Kaisha Ltd., Osaka, Japan), according to the manufacturer’s instructions. A 160-µL HVJ envelope vector suspension was centrifuged at 10,000 x g for 5 min. The pellet was re-suspended in 10 µL rgpA DNA vaccine (16 µg/µL) and 80 µL bovine serum albumin (BSA) solution. After the addition of 2% Triton X-100 (8 µL), the mixture was centrifuged at 10,000 x g for 5 min. The pellet was finally re-suspended in 80 µL BSA solution. The mice were nasally immunized with rgpA-HVJ-E vector (20 µg/10 µL/mouse) at wks 0, 1, 2, 3, 4, 5, 6, and 7. Vaccination for investigation of the kinetics of the antibody response and evaluation of the protective effect of the vaccination described below was performed independently.

ELISA and Immunoblot
Serum and saliva samples were obtained from the mice on days 0, 7, 14, 21, 28, 35, 42, and 49. Anti-rgpA antibodies in the serum and saliva were detected by enzyme-linked immunosorbent assay (ELISA) as previously described (Yonezawa et al., 2001). A 20-µg sonic extract of P. gingivalis ATCC33277 was coated onto flat-bottomed polystyrene microplates (Corning, Corning, NY, USA), and the wells were blocked. Duplicate serial two-fold-diluted samples were applied to the wells and incubated at 37°C for 60 min. After wells were washed, peroxidase-labeled goat antibody against mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgA (ICN Biomedicals, Inc., Aurora, OH, USA) was applied. After development, absorbance at 405 nm was measured with a microplate reader (Bio-Rad Laboratories). The endpoint titer for antigen-specific immunoglobulins was defined as the last dilution giving an optical density at 405 nm of > 0.15. Specificity of salivary sIgA was confirmed by immunoblot analysis, with each recombinant fusion protein of the Rgp domain expressed by pET32Xa/LIC (Novagen, Gibbstown, NJ, USA), as described previously (Yonezawa et al., 2001). These consisted of the histidine tag protein and each domain. The deduced molecular masses of the histidine-tag (17388.5) of pET32Xa/LIC and catalytic subunits, HGRP44 and HGRP 15-27, were 71258.3, 62058.8, and 78729.4, respectively.

P. gingivalis Challenge Studies
To evaluate the protective effect of the vaccination against P. gingivalis, we infected the immunized mice with P. gingivalis as described previously (Baker et al., 1994), with minor modifications. The BALB/c mice were separated into 4 groups: a non-immunized group (n = 10), a group immunized with the rgpA DNA vaccine via the skin of the abdomen by a Gene Gun (n = 5), a group immunized intranasally with rgpA-HVJ-E vector (n = 5), and a group immunized with pVAX1 (Invitrogen, Carlsbad, CA, USA) alone (n = 5). The serum IgG and salivary sIgA antibody titers were evaluated at 4 to 6 wks as described above. When the antibody titers of the mice immunized by Gene Gun and intranasally with rgpA-HVJ-E vector reached a plateau, the mice were challenged with P. gingivalis. Following immunization, the mice were given 5 mg each of kanamycin and ampicillin by gavage, once a day for 4 days. This was followed by a three-day antibiotic-free period. They were then challenged with approximately 1 x 109 CFUs of live P. gingivalis ATCC33277 suspended in 100 µL of 2% carboxymethylcellulose via a feeding needle, except for the 5 non-immunized mice. These 5 non-infected and non-immunized mice were used to determine the baseline value from the cementum-enamel junction (CEJ) to the alveolar bone crest (ABC) in normal mice. The challenge was carried out 3 times at two-day intervals. Forty-two days after the last gavage, the mice were killed. We then repeated the experiment to confirm reproducibility, and performed statistical analysis using the pooled data from both experiments.

Measurement of Alveolar Bone Loss
Alveolar bone loss was assessed at defined landmark sites on the maxillary molars of each mouse as described previously (Gonzalez et al., 2003). We performed measurements (14 sites) of each skull from the CEJ to the ABC with a stereomicroscope. Measurements were made under a dissecting microscope fitted with a video image-maker measurement system, MS-803 (MORITEX Co., Tokyo, Japan), standardized to give measurements in millimeters.

Statistical Analysis
We used a one-way ANOVA followed by a Student-Newman-Keuls test to make multiple comparisons between the relative protective effects of the DNA vaccinations against bone loss. The differences in antibody levels among the groups were also evaluated with a one-way ANOVA, followed by a Student-Newman-Keuls test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Responses of Immunoglobulins to P. gingivalis
Specific IgGs were clearly induced in both groups of BALB/c mice inoculated with the rgpA DNA vaccine, either by Gene Gun or intranasally (Fig. 1AGo). Following immunization, the responses in both groups reached plateaus on day 42. The titers of IgGs at day 49 in both immunized groups were elevated (p < 0.01), with the Gene Gun group yielding slightly lower titers than the intranasal group. The specificities of serum IgGs to recombinant RgpA domains were confirmed as described previously (Yonezawa et al., 2001).


Figure 1
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  		Figure 1. Induction of P. gingivalis-specific serum IgG and salivary sIgA antibodies in mice immunized with rgpA DNA vaccine via abdominal skin by Gene Gun (2.5 µg/mouse) and mice immunized nasally with the rgpA-HVJ envelope vector (20 µg/mouse). rgpA DNA vaccine was injected a total of 7 times as indicated by arrows. (A) Serum IgG titers of mice against sonicates of P. gingivalis were determined on days 0, 7, 14, 28, 35, 42, and 49 after primary immunization (N = 15). Serial dilutions were used to measure endpoint titers (> 0.15). Results are means ± standard deviations of log2 ELISA antibody titers. Variation in endpoint titers was within a two-fold serial dilution. *p < 0.01, against naïve mice; **p < 0.01 against Gene Gun-immunized mice by one-way ANOVA, followed by the Student-Newman-Keuls test. (B) Salivary sIgA titers against sonicates of P. gingivalis were determined on days 0, 7, 14, 28, 35, 42, and 49 after primary immunization. Serial dilutions were used to measure endpoint titers (> 0.15). Results are means ± standard deviations of log2 ELISA antibody titers (N = 15). Variation in endpoint titer was within a two-fold serial dilution. *p < 0.01 against naïve mice and Gene Gun-immunized mice by one-way ANOVA, followed by the Student-Newman-Keuls test.

 
Intranasal immunization with rgpA DNA vaccine induced P. gingivalis-specific sIgA responses in the saliva, with this response reaching a plateau on day 42 (p < 0.01, Fig. 1BGo). In contrast, no sIgA production was observed in the Gene Gun group.

The specificity of salivary sIgA against each recombinant RgpA domain is shown in Fig. 2Go. It reacted to the catalytic domains of RgpA, HGRP 44, and HGRP 15–27. The molecular masses corresponded well with the deduced molecular masses of the recombinant fusion proteins of each domain with a histidine-tag of pET32Xa/LIC.


Figure 2
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  		Figure 2. Specificity of salivary sIgA against RgpA domains. Recombinant catalytic domain of RgpA (RgpA Cat, lane 1), 44-kDa adhesion hemagglutinin domain (HGRP 44, lane 2), and 15-, 17-, and 27-kDa hemagglutinin domains (HGRP 15–27, lane 3) with histidine tags were separated by SDS-PAGE and transferred to PVDF membrane. Blotted membrane was blocked with phosphate-buffered saline containing 3% bovine serum albumin, reacted with peroxidase-conjugated anti-mouse IgA and developed. Blotted lanes 1, 2, and 3 were probed with saliva from mice immunized nasally with rgpA-HVJ envelope vector.

 
The profiles of the IgG subclasses were the same in both groups, with the most prevalent subclass being IgG1, followed by IgG2b (p < 0.01, Fig. 3Go).


Figure 3
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  		Figure 3. P. gingivalis-specific IgG subclass responses against sonicates of P. gingivalis. Immunization groups of BALB/c mice immunized with rgpA DNA vaccine via abdominal skin by Gene Gun (2.5 µg/mouse) or immunized nasally with rgpA-HVJ envelope vector (20 µg/mouse). IgG subclass responses were assessed on day 49. Results are means ± standard deviations of log2 ELISA antibody titers (N = 15). Variation in endpoint titers was within a two-fold serial dilution. One-way ANOVA for repeated measurements was used for inter-group comparisons. The Student-Newman-Keuls test was used for multiple comparisons.

 
Immunization with rgpA DNA Vaccine Protects against Alveolar Bone Loss Incurred by Infection with P. gingivalis.
The level of alveolar bone loss was assessed at 42 days after challenge with P. gingivalis ATCC33277. The infected mice showed significantly greater maxillary molar alveolar bone loss than did the uninfected control mice (p < 0.01, Fig. 4Go). Both the Gene Gun group and the intranasal group were protected from alveolar bone loss (p < 0.01), although the magnitude of protection was greater in the latter (p < 0.01).


Figure 4
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  		Figure 4. Levels of alveolar bone loss elicited following P. gingivalis oral challenge. BALB/c mice immunized with rgpA DNA vaccine via abdominal skin by Gene Gun (2.5 µg/mouse), mice immunized nasally with rgpA-HVJ envelope vector (20 µg/mouse); control group consisted of age-matched, non-vaccinated mice and pVAX1 immunized mice orally challenged with P. gingivalis ATCC 33277 (Pg). At 42 days after oral challenge, all mice were killed. Linear measurements (n = 14 sites) were obtained from the mean of the maxillary molars in each mouse, and pooled linear measurements (in millimeters) were calculated for each group (N = 20 for non-infected naïve mice, P. gingivalis-infected naïve mice, and P. gingivalis-infected HVJ envelope vector-immunized mice; N = 15 for P. gingivalis-infected Gene Gun-immunized mice; N = 15 for pVAX1 immunized with HVJ envelope vector). Data are shown as mean ± standard deviation. One-way ANOVA for repeated measurements was used for inter-group comparisons. The Student-Newman-Keuls test was used for multiple comparisons (*p < 0.01). CEJ = cemento-enamel junction, ABC = alveolar bone crest.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Gingipain is a major pathogen of P. gingivalis (Potempa et al., 2003). We found that both serum IgG and sIgA production was induced in mice intranasally immunized with rgpA DNA vaccine by an HVJ envelope vector; in contrast, immunization via the abdominal skin by Gene Gun induced serum IgG alone. These results suggest that intranasal administration of DNA vaccine by an HVJ envelope vector can induce a stronger response in the mucosal immune system. We found that the levels of induced IgG against RgpA were almost the same in both groups. These results indicate that intranasal rgpA DNA vaccine immunization is a more effective procedure for inducing serum and salivary antibody responses in the absence of any adjuvant. Kawabata et al.(1999) demonstrated that injection of fimbriae DNA vaccine into salivary glands induced salivary IgA and serum IgG. In our results, the level of salivary IgA was almost the same as that reported in their data; however, the serum IgG titer was lower. It is possible that this variation in IgG titers was a result of the different immunization routes used.

Analysis of each IgG subclass level against P. gingivalis revealed that immunization with rgpA DNA vaccine via either the abdominal or the intranasal route strongly induced production of IgG1 and IgG2b. This DNA vaccine offers the advantage of inducing both humoral and cell-mediated immune responses. Our results suggest that immunization with rgpA DNA vaccine preferentially induced antibody via Th2-type responses. Another group has also reported a protective effect resulting from serum IgG response to P. gingivalis (Gibson et al., 2004). Analysis of these data, taken together, indicates that the induction of antibody via Th2-type response plays an important role in protecting against infection by P. gingivalis.

The Th1-type response has been associated with destruction of periodontal tissue and bone resorption (Takeichi et al., 2000; Taubman and Kawai, 2001). In an earlier study, we found that rgpA DNA vaccine induced a reduction in Th1-type cytokines such as IFN-{gamma}, and a protective effect in mice challenged with a lethal dose of P. gingivalis (Yonezawa et al., 2005). It is possible that the protection afforded by vaccination with the rgpA DNA vaccine resulted from a reduction in the Th1-type response. However, the Th1-type reaction plays an important role in protection against acute infection (Spellberg and Edwards, 2001). To clarify the relationship between reduction in Th1-type response and protective response to vaccination, further analysis is required to investigate the Th1/Th2 balance in vaccinated mice during the inflammation process.

Numerous clinical studies in patients with chronic periodontitis have demonstrated high antibody levels against P. gingivalis in serum and gingival crevicular fluid (Tew et al., 1985; Ebersole et al., 1986; Inagaki et al., 2003). However, few reports have investigated the protective effects of immunoresponses against periodontal disease in humans. Booth et al.(1996) found that passive immunization with monoclonal antibody against P. gingivalis inhibited recolonization. We previously reported that rgpA DNA vaccine induced a protective effect against P. gingivalis infection in mice (Yonezawa et al., 2001). Another study (Genco et al., 1998) showed that a peptide domain on R gingipain had a protective effect against P. gingivalis infection in mice. Mice immunized with RgpA polypeptides were protected from P. gingivalis-elicited oral bone loss, with this antigen also eliciting a potent IgG response (Gibson et al., 2001). In addition, IgG against purified RgpA also showed a protective effect, enhancing PMN-mediated bacterial killing of P. gingivalis strains (Nakagawa et al., 2001). When these findings are considered together with the present results, it seems clear that IgG can exert a preventive effect against components of P. gingivalis.

Intranasally immunized mice were better protected against bone loss than those immunized with the Gene Gun, suggesting that sIgA offers a protective advantage over high IgG antibody levels alone. Specific sIgA against Streptococcus mutans has a protective effect against colonization by the pathogens in the oral cavity (Saito et al., 2001). It is possible that sIgA reduces colonization by P. gingivalis. However, in this study, it was not possible to evaluate the inhibition of oral colonization, since P. gingivalis is not a resident of the oral cavity in the mouse; furthermore, exposure to such high numbers of this microorganism here may have induced bone loss. Such factors must be taken into account in evaluation of the reduction of colonization. Therefore, further study is required in a suitable animal model to clarify the effect of sIgA.

The results of this study suggest that intranasal immunization with an rgpA DNA vaccine offers strong potential in the prevention of periodontal disease. This research offers a new approach to the clinical prevention of periodontal disease using a combination of rgpA DNA vaccine and the HVJ envelope vector system.


   ACKNOWLEDGMENTS
 
This study was supported by grants to Kazuyuki Ishihara (16591837) and Katsuji Okuda (14370608) from the Ministry of Education, Science, Sport, Culture and Technology (MEXT) of Japan, by Grant HRC7 from the Oral Health Science Center of Tokyo Dental College, and by a "High-Tech Research Center" Project for Private Universities matching fund subsidy from MEXT, 2000–2005. We thank Mr. Jeremy Williams for editing this manuscript.

Received September 23, 2005; Last revision December 3, 2006; Accepted January 1, 2007


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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