REVIEW
Gingipains as candidate antigens for Porphyromonas
gingivalis vaccine
Taneaki Nakagawa, Atsushi Saito1, Yasuo Hosaka and Kazuyuki Ishihara2
Department of Dentistry and Oral Surgery, School of Medicine, Keio University, 1Department of Periodontics and
2Department of Microbiology, Tokyo Dental College, Tokyo, Japan(Received for publication on February 6, 2003)

Abstract. Porphyromonas gingivalis (P. gingivalis), a gram-negative anaerobe, is involved in thepathogenesis of periodontal disease, and is found frequently in the subgingival ﬂora in patients withperiodontitis. This organism possesses a variety of virulence factors including lipopolysaccharide, capsular material, ﬁmbriae and proteases (enzymes). Among the P. gingivalis antigens, enzymes such as
Arginine-speciﬁc gingipains (RgpA, RgpB) and lysine-speciﬁc gingipain (Kgp) have been studied fortheir ability to induce biologically signiﬁcant antibodies. This review summarizes recent information on
the gingipains and their possible application in the development of an anti-P. gingivalis vaccine. (Keio J
Med 52 (3): 158–162, September 2003)
Key words: Porphyromonas gingivalis, gingipain, vaccine, periodontal disease

Introduction

Pathogenicity of P. gingivalis

Two major form of periodontal disease are common
in man and other animals, gingivitis and periodontitis.
Seventy to eighty percent of the adult population sufferfrom this disease. Both forms are characterized by inﬂammation of the gingival tissue in response to thepresence of microbial plaque on the teeth. Gingivitis
can be distinguished from the various types of periodontitis by absence of surrounding tissue destruction.
Gingival inﬂammation can be eliminated in gingivitis byplaque removal and improved dental hygiene. In thecase of periodontitis, chronic inﬂammation induced byplaque is accompanied by the formation of characteristic gingival lesions. When the periodontal ligament andbone supporting affected teeth are partially destroyed,
the gingival epithelium becomes detatched from theteeth, and pathologic periodontal pockets are formed.
The disease is caused by a group of predominantly gramnegative anaerobic bacterial species in dental plaque.
Principal among these are Porphyromonas gingivalis(P. gingivalis) and Bacteroides forsythus in case of adultperiodontitis and Actinobacillus actinomycetemcomitansin localized juvenile periodontitis. This review mainly
focuses on P. gingivalis.

P. gingivalis, a gram-negative anaerobe, is involvedin the pathogenesis of periodontal disease, found more
frequently and as a higher population of the subgingival
ﬂora in patients with periodontitis than in control subjects.1,2 This microorganism easily survives in a hostileenvironment by successfully evading host antimicrobial
defenses, utilizing variety of virulence factors like ﬁmbriae, a polysaccharide capsule and lipopolysaccharide,
hemagglutinin and hemolyzing activity, release of toxic
products of metabolism, outer membrane vesicles andnumerous enzymes.3–6 The ﬁmbriae of this microorganism not only play a role in colonization of the microorganism7 but also activate cytokine production likelipopolysaccharide.8,9 The activation of host defense
and induction of the inﬂammatory cytokines such as
interleukin 1 and tumor necrotizing factor a play animportant role in bone absorption.10
Signiﬁcant interest has been shown in the ‘‘trypsinlike activity’’ to clarify the pathogenesis of this microorganism. These enzyme downregulate polymorphonuclear neutrophils, degrade extracellular protein
and bioactive peptides such as C5, prekallikrein andkininogen. P. gingivalis has several cysteine proteases

Reprint requests to: Dr. Taneaki Nakagawa, Department of Dentistry and Oral Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan, e-mail: tane@sc.itc.keio.ac.jp158Keio J Med 2003; 52 (3): 158–162

called as gingipains. Gingipains are classiﬁed into twogroups based on substrate speciﬁcity. Gingipains R(RgpA and RgpB) cleaves proteins after arginine residue and are encoded by two similar genes, RgpA and
RgpB, while gingipain K (Kgp) cleaves proteins after
lysine residue. RgpA together with RgpB, account for
all trypsin-like activity of P. gingivalis. Kgp plays animportant role in Fe acquisition by binding to hemoglobin. These properties strongly affect the virulence of
this microorganism.11
Immune Response to P. gingivalis
Humoral responses to P. gingivalis have been foundin high frequency, and our data have shown that theserum IgG antibody levels to P. gingivalis correlatedwith the presence of P. gingivalis in periodontal pockets, but not with other bacteria tested.1,12
However, the IgG titers to P. gingivalis were merely
related to the ‘number’ of microﬂora present. Therefore, to assess the biological signiﬁcance of the antibody, we investigated IgG avidity to P. gingivalis.13
The study showed that patient sera with high IgG titers
demonstrated low values for avidity, suggesting that
IgG responses in patients with adult periodontitis play alimited role in colonization inhibition or eliminationof P. gingivalis. The data also showed that periodontally healthy individuals have highly functional
antibodies, which may protect them against P. gingivalis colonization. Also our data indicated periodontitis
patients mount a humoral immune response to theantigens of P. gingivalis following therapy, and these
antibodies may be protective. Recently, several reports
indicate that infection with periodontopathic bacteria
is associated with systemic diseases such as endocarditis, cardiovascular disease, and aspiration pneumonia.14–17 Although the precise role of a speciﬁc IgG
antibody in the etiology of the disease is still uncertain,
vaccine directed against antigens of the infecting bacteria may be effective in preventing and arresting the
progresses of these diseases, and there has been considerable interest in vaccine development for periodontitis.
Another aspect for the requirement of vaccine development for periodontal disease is as follows. Efforts
to prevent or arrest the progress of periodontitis havetraditionally been directed in a major part toward removal and control of deposit of microorganism on thesurface of teeth and elimination of infectious microorganisms. Treatment requires specialty care which is
expensive and of limited availability. It can be painful
and frequently unsuccessful, resulting in recurrence ofor a contribution to the destructive process. More effective approaches for the prevention and control of
periodontitis are needed.

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Using the ligature-induced periodontitis model in
Macaca fascicularis, Persson et al.18 have shown that
immunization using a vaccine containing whole killed P.
gingivalis as antigen signiﬁcantly inhibits the onset andprogress of disease as measured by radiographic assessment of alveolar bone loss. Booth et al.19 reported
that the monoclonal antibodies against P. gingivalis
whole cells prevent the re-colonization of P. gingivalis
into the gingival crevice. The results indicated that
antibodies against certain parts of the P. gingivalis surface antigen play a protective role. The next step invaccine development is the identiﬁcation and testing ofa puriﬁed P. gingivalis component that can induce protection against infection by this microorganism.
Gingipains as Target Antigens for Vaccine
In searching for the appropriate antigen, we andothers have examined a group of cell surface carbohydrates designated as K-antigens,20,21 lipopolysaccharides,22,23 and various proteins including ﬁmbriae,24,25
the 53-KDa and 67K-Da cell surface proteins,26 hemagglutinin27 and cysteine proteases referred to as gingipains.28–32 Of the P. gingivalis components studied todate, the gingipains have shown the highest potential
for use as vaccine antigens. Gingipains are present inlarge quantities on the cell surface of P. gingivalis, andthey can signiﬁcantly contribute to the virulence exhibited by this species.
The mature form of RgpA possesses both a catalytic
domain and a hemagglutinin domain, while RgpB possesses only a catalytic domain. The hemagglutinin domain plays a role in the adherence of this microorganism to erythrocytes of this microorganism. There is ahigh degree of homology between the catalytic domainsof RgpA and RgpB at both the DNA and protein levels, while the hemagglutinin domain of RgpA is similar
to the P. gingivalis hemagglutinin domain of Kgp. In
vitro studies have shown that gingipains are able todegrade collagen and ﬁbronectin, inactivate proteaseinhibitors, degrade immunoglobulins, and facilitate ironacquisition. Furthermore, they are able to destroy hostcoagulation cascade proteins, degrade complement, anddigest various cytokines.31
Arginine-speciﬁc Gingipains (RgpA) and lysinespeciﬁc gingipain (Kgp), enzymes produced by P. gingivalis, may be candidates for an anti-P. gingivalis vaccine. We initiated a study to determine whether RgpA
and Kgp have opsonic target sites and whether thesesites are available and accessible on intact P. gingivalis cells.32 Rabbits were immunized and IgG fractions were isolated from preimmune and immune
sera. Functional characteristics of the antibodies wereassessed by determining antibody titers by ELISA,
generating Western blots, and measuring of the anti-

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body enhancement of P. gingivalis opsonization, phagocytosis and killing by polymorphonuclear leukocytesof intact cells of P. gingivalis representative of thefour serotypes. Both RgpA and Kgp induced high titersof IgG antibody. Anti-RgpA and anti-Kgp antibodies
bound to both RgpA and Kgp demonstrating a large
proportion of shared antigenic epitopes. The two antibodies bound equally well to all four P. gingivalis serotypes compared to preimmune IgG. The immunoblot
patterns of binding of the two antibodies to RgpA andKgp and to sonicates of the four P. gingivalis serotypes
were virtually identical (Fig. 1). Both proteins induced
antibodies that signiﬁcantly enhanced opsonization asassessed by chemiluminescence (Fig. 2). Both antibodies signiﬁcantly enhanced PMN-mediated bacterial
killing of the four P. gingivalis serotypes (Fig. 3). These
data indicate these two proteins appear to be potential
candidate antigens for an anti-P. gingivalis vaccine.
We have similar data on RgpB, which possesses onlya catalytic domain. The data indicate that RgpB also
appear to be potential candidate antigen for anti-P.
gingivalis vaccine (unpublished data).
It is not clear yet which gingipains (RgpA, RgpB or
Kgp) would be the best candidate as a periodontal vac-

Nakagawa T, et al: Gingipains for periodontal vaccine

Fig. 1

Schematic comparison of gingipains.

cine. Recent studies have mostly used the mouse or rat
model. Booth et al. reported that passive immunization
using an anti-P. gingivalis monoclonal antibody prevented the re-colonization of P. gingivalis in humanvolunteer19. Their additional report indicated that the
monoclonal antibody recognized the hemagglutinin domain of RgpA.33,34 Gibson et al.35 examined the abilities of RgpA and RgpB to elicit protection against
P. gingivalis-mediated oral bone loss in a murine oral

Fig. 2 Opsonic activity measured as chemiluminescence (CL) and reported as millivolts for anti-HRgpA IgG (A) and anti-Kgp IgG (B) antibodies to P. gingivalis strains 33277, 381, W50 and A7A1-28. The uppermost set of curves was from incubations containing active complement
and lower most contained heat-inactivated complement. The bold lines are the results for incubations containing immune IgGs and ﬁne lines for
incubations containing preimmune IgGs. (Reproduce from Nakagawa T, et al: Oral microbiology and Immunology 2001; 16: 202–211, Copyright
6 (2001), with permission from Blackwell Munksgaard)

Keio J Med 2003; 52 (3): 158–162

Fig. 3 PMN-mediated phagocytosis and killing of each of the four
serotype strains of P. gingivalis. In the presence of preimmune IgG(negative control), anti-whole cell IgG (positive control) and antiHRgpA IgG and anti-Kgp IgG reported as the percentage, with standard error, of viable bacteria remaining at the end of the incubation
period. (Reproduce from Nakagawa T, et al: Oral microbiology and
Immunology 2001; 16: 202–211, Copyright 6 (2001), with permission
from Blackwell Munksgaard)

challenge model. Mice immunized subcutaneously withheat killed RgpA or RgpB possessed elevated levels of
P. gingivalis-speciﬁc immunoglobulin G; however only
the animals immunized with RgpA were protected frommaxillary bone loss. They concluded that immunization with RgpA stimulates the production of hemagglutinin domain-speciﬁc antibodies, which contribute to
the prevention of P. gingivalis-mediated periodontal
disease. This report suggests that antibody responses
against hemagglutinin domain contribute to the prevention of P. gingivalis-mediated periodontal disease.
Yonezawa et al.36 reported immunization of mice with
the RgpA DNA vaccine resulted in the production of
antibodies. The vaccine strongly induced antibodies tothe hemagglutinin domain and weakly to the catalytic
domain. A protective effect against P. gingivalis virulence was observed in the mouse lesion model. However, Inagaki et al.37 reported that the sera of patientswith periodontitis showed high IgG titers against thehemagglutinin domain but not to the catalytic domain.
They suggested that the antibodies against the hemagglutinin domain did not have protective immunity.
Taken together, some part of the antibody against thehemagglutinin domain antibody seem to be protective
and others do not. Further investigation is required toclarify the epitope that induces protective antibodies.
On the other hand, the importance of the antibody
against the catalytic domain has stated in several
reports. Inagaki et al.37 reported that the levels of antibodies to the catalytic domain are the same between

161

healthy subjects and periodontitis patients. They suggested the antibodies to the catalytic domain are important in the prevention of P. gingivalis infection.
Genco et al.38 reported that an antibody induced by the
peptide of the gingipain R catalytic domain showed aprotective effect against P. gingivalis infection in themouse model. O’Brien-Simpson et al.39 also reported
that the peptide designed from the catalytic domain
induced a protective effect against P. gingivalis infection in the mouse model. Kuboniwa et al.40 reported
that an antibody against the Kgp catalytic domain DNA
vaccine reacted to Rgp and that the vaccine induced aprotective effect against a P. gingivalis challenge in themurine model. These reports suggested the importanceof the anti-catalytic domain antibodies of gingipains,
and that induction of the antibodies against the catalytic
domain is protective against P. gingivalis infection.
Further analysis is required to clarify the low antibody
titers in periodontal patients and the protective effect ofantibodies against the catalytic domain.
The reports also suggested some epitope in catalytic
and hemagglutinin domain elicited a protective effectagainst P. gingivalis infection. P. gingivalis is known toinvade epithelial cells. A recent report suggested thatthe hemagglutinin domain was implicated in the nuclear
targeting of this protease.41 The antibodies against gingipain are important in the invasion. The evaluatedvaccines are mainly peptide and DNA vaccines. They
induce different types of immunoresponses. DNA
vaccine induced both cellular immunity and humoral
immunity. On the other hand, a peptide vaccine induces humoral immunity. Further analysis is required toevaluate at which stage these immunoresponses prevent
P. gingivalis infection. Evaluation of the epitopes, immunization rates and the effective adjuvant is in progress in our laboratories.
In summary, recent data indicate that the gingipains(RgpA and Kgp) of P. gingivalis are potential candidates for a vaccine that could be used for prevention of
P. gingivalis-mediated periodontal disease.
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