Department of Medicine, Section of Infectious
Diseases, Boston University School of Medicine, Boston,
Massachusetts 02118
Received 2 May 2001/Returned for modification 18 June 2001/Accepted 13 September 2001
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TEXT |
Periodontal diseases in the past
have been characterized as a broad group of infections with multiple
bacterial etiologies (19, 26, 27). However, recent
evidence strongly suggests that the primary pathogen responsible for
adult progressive periodontal disease is Porphyromonas
gingivalis. Following colonization of the gingival crevice,
P. gingivalis initiates inflammation of the gingivae,
destruction of periodontal tissue, loss of alveolar bone, and in severe
cases, exfoliation of teeth. This organism possesses a variety of
virulence factors, including fimbriae, lectin-like adhesins, capsular
polysaccharide, lipopolysaccharide, hemagglutinins, and hemolysins, as
well as numerous proteolytic enzymes (10, 12, 20, 31).
Some of the proteases, the gingipains, a group of cysteine proteases
produced by P. gingivalis, have received considerable
attention. Gingipains are classified into two groups based on substrate
specificity. Gingipains R (RgpA and RgpB) cleave proteins after
arginine (R) residues and are encoded by two similar genes,
rgpA and rgpB, while gingipain K (Kgp) cleaves
proteins after lysine (K) residues (21, 28). The mature
form of RgpA possesses both a catalytic domain and a hemagglutinin
domain, while RgpB possesses only a catalytic domain (30).
There is a high degree of homology between the catalytic domains of
RgpA and RgpB at both the DNA and protein levels, while the
hemagglutinin domain of RgpA is similar to both P. gingivalis hemagglutinin HagA and the tla gene product
(9). In vitro studies have shown that gingipains are able
to degrade both collagen and fibronectin, inactivate protease
inhibitors, degrade immunoglobulins, and facilitate iron acquisition
(10, 25, 29). Furthermore, they are able to destroy host
coagulation cascade proteins, degrade complement, and digest various
cytokines (3, 5, 10, 13-15).
Several studies have demonstrated that immunization of animals with
relevant P. gingivalis antigens, including fimbriae and porphypain 2 (gingipain K), as well as HagA and HagB, may provide protection against subsequent P. gingivalis challenge in
various animal models (6, 7, 16, 22). Genco et al.
(9) demonstrated that treatment of P. gingivalis with various protease inhibitors prior to challenge of
mice significantly reduced morbidity and mortality compared to the
morbidity and mortality of animals challenged with untreated P. gingivalis. More recently, it was observed that mice immunized
with RgpA or RgpB produced increased levels of immunoglobulin G (IgG)
and were protected from subsequent P. gingivalis challenge
when a chamber infection model was used (9). These observations correlate well with human studies, which have shown that
patients with rapidly progressive periodontal disease possess elevated
levels of serum antibody to the hemagglutinin domain of P. gingivalis RgpA (23).
Recently, Baker et al. (2) demonstrated that oral
challenge of mice with P. gingivalis stimulated oral bone
loss and that the observed bone loss occurred in a site-specific
manner. Furthermore, it appears that oral bone loss is linked to T-cell
activation (1). In the present study we assessed whether
the arginine gingipains could be vaccine candidates for prevention of
oral bone loss in a murine model.
P. gingivalis and gingipain preparation.
P.
gingivalis A7A1-28 (obtained from Pamela Baker, Bates College,
Lewiston, Maine) was grown anaerobically on anaerobic blood agar plates
supplemented with hemin and menadione (BBL, Cockeysville, Md.).
Bacterial growth was collected from plates and suspended in
sterile phosphate-buffered saline (pH 7.2), and the optical density at
660 nm was adjusted to either 3.0 (approximately 1 × 1010 CFU/ml) for gavage of mice or 0.3 for
immunizations and enzyme-linked immunosorbent assay (ELISA) plate
coating. Heat-killed P. gingivalis was prepared by
incubating 1 ml of cells, adjusted to an optical density at 660 nm of
0.3 in phosphate-buffered saline, at 60°C for 5 min, and an aliquot
of the preparation was plated to confirm the loss of P. gingivalis viability. Gingipains RgpA and RgpB were isolated and
purified as previously described (9) and were kindly
provided by Jan Potempa (Jagiellowian University, Cracow, Poland).
Mouse immunization and challenge studies.
A stainless steel
wire chamber was surgically implanted under the skin of each 6- to
8-week-old BALB/c mouse (Jackson Laboratories, Bar Harbor, Maine)
(8). Preimmune chamber fluid samples were collected from
each mouse, and the animals were separated into groups (eight animals
per group), including a nonimmunized group and groups that were
immunized subcutaneously (100 µl/injection) with Freund's complete
adjuvant or with heat-killed P. gingivalis or adjuvant
containing either RgpA and RgpB (100 µg/injection). The animals then
received weekly booster doses for 3 weeks with the respective antigen
suspended in incomplete adjuvant (Fig. 1). Prior to each immunization,
chamber fluid samples were collected from each mouse, pooled by group,
and stored frozen until P. gingivalis-specific IgM or IgG
ELISAs were performed. Following immunization mice were challenged
orally three times (approximately 1 × 109 CFU per
challenge) with P. gingivalis A7A1-28 by the method of Baker
et al. (2). P. gingivalis colonization of
maxillary molars of mice was assessed with sterile paper points
(2). Forty-two days after gavage, the mice were
sacrificed, the heads were collected, and each skull was cleaned with
hot water, 3% hydrogen peroxide, and 0.1% hypochlorite and was
stained with 1% methylene blue. Seven linear (millimeter) and three
area (square millimeter) measurements were obtained from the left and
right sets of maxillary molars from each skull by using a
stereomicroscope with an onscreen computer-aided measurement package
(Image-Pro Plus V 3.0; Media Cybernetics, Silver Spring, Md.). These
experiments were performed twice for a total of 16 animals per group.
Means ± standard errors of the means were determined for all
linear and area measurements. The Mann-Whitney nonparametric test was
performed to compare groups (InStat V 2.0; Graphpad Software, San
Diego, Calif.). Significant differences between groups were determined
by using a P value of <0.05.
Immunization of mice with RgpA and RgpB stimulates P. gingivalis-specific antibodies. (i) ELISA analysis.
ELISAs
were performed with all chamber fluid samples to detect the amounts of
P. gingivalis-specific IgM and IgG in each sample. Immunol
4Hbx ELISA plates (Dynex, Chantilly, Va.) were coated with 50 µl of
formaldehyde-fixed P. gingivalis A7A1-26 in
carbonate-bicarbonate buffer (pH 9.6) per well and were blocked with
2% bovine serum albumin, serial twofold dilutions of chamber fluid
samples were added to the wells, and the plates were incubated
overnight. The wells were washed, and 100-µl portions of either goat
anti-mouse IgM- or IgG-alkaline phosphatase conjugate (Sigma, St.
Louis, Mo.) were added to the wells. The plates were developed with
p-nitrophenyl phosphate and were read at 405 nm. The
concentration of P. gingivalis-specific IgG was determined
from a murine IgG standard curve ELISA preparation that was run in
parallel with test samples. Immunization of mice with heat-killed
P. gingivalis, RgpA, or RgpB resulted in maximal levels of
P. gingivalis-specific IgM within 3 weeks (data not shown).
The level of P. gingivalis-specific IgG present in chamber fluid samples from mice that were immunized with heat-killed P. gingivalis was robust (Fig. 2).
Immunization with RgpA also resulted in a potent P. gingivalis-specific IgG response similar to that observed after
immunization with heat-killed P. gingivalis (Fig. 2).
Interestingly, animals immunized with RgpB produced only low levels of
P. gingivalis-specific IgG. Nonimmunized animals, as well as
animals given adjuvant alone, produced negligible amounts of P. gingivalis-specific IgM or IgG.
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FIG. 1.
Time line of events during immunization-oral challenge
experiments. Groups of BALB/c mice received immunizations (Im, B1 to
B3), had their sera collected (C1 to C5), and were orally gavaged with
P. gingivalis A7A1-28 (G1 to G3). After a 42-day oral bone
loss period, mice were sacrificed.
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(ii) Immunoprobe analysis.
To determine which polypeptides of
RgpA and RgpB were recognized by chamber fluid samples from each group
of immunized animals, P. gingivalis whole-cell lysates,
RgpA, and RgpB were separated on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels
(18). When purified RgpA and RgpB are denatured and
reduced and then separated on a SDS-PAGE gel, five bands, including a 45-kDa band (catalytic domain) and 44-, 27-, 17-, and 15-kDa bands (four polypeptides of the hemagglutinin domain), are resolved or a
single 50-kDa band is resolved (30). The blots were either stained with Simply Blue SafeStain (Invitrogen, Carlsbad, Calif.) (Fig.
3A) or transferred to polyvinylidene
difluoride membranes, probed with chamber fluid samples, and developed
with goat anti-mouse IgG-alkaline phosphatase conjugate and with
phosphatase substrate (Bio-Rad). As expected, week 4 chamber fluid
samples obtained from untreated animals failed to recognize P. gingivalis whole-cell lysates, RgpA, or RgpB on immunoblots (Fig.
3A). Chamber fluid samples collected from animals immunized with
heat-killed P. gingivalis recognized a broad range of
high-molecular-mass antigens and several lower-molecular- mass bands in
the P. gingivalis whole-cell lysate (Fig. 3B). Furthermore,
these chamber fluid samples recognized each of the five polypeptide
bands of RgpA, as well as RgpB (Fig. 3B). Chamber fluid samples
obtained from mice immunized with RgpA recognized all polypeptides of
the RgpA complex, as well as RgpB. Additionally, these chamber fluid
samples recognized a 44-kDa band in the P. gingivalis
whole-cell lysate corresponding to the high-molecular-mass
hemagglutinin domain of RgpA (Fig. 3C). Chamber fluid from mice
immunized with RgpB recognized purified RgpB but failed to recognize
any domain of RgpA or any antigens in P. gingivalis whole-cell lysates (Fig. 3D).
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FIG. 2.
Quantification of P. gingivalis-specific IgG
levels in week 4 chamber fluids of BALB/c mice following immunization.
Chamber fluids were collected from nonimmunized (None) mice or from
animals that received adjuvant only (Adj), heat-killed P. gingivalis (HK Pg), RgpA, or RgpB, and the levels of P. gingivalis-specific IgG were determined. The data are means based
on two experiments (n = 16 mice), and the error bars
indicate standard deviations.
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Immunization of mice with RgpA but not RgpB protects the animals
from P. gingivalis-induced bone loss.
Immunized
animals were subsequently orally challenged with P. gingivalis, and the level of oral bone loss was assessed 42 days
after the challenge (Fig. 1). Since oral
bone loss during rapidly progressive periodontal disease occurs in both
an overall manner and a site-specific manner, seven linear and three
area measurements were obtained for each set of maxillary molars per animal. The overall bone loss in each group was determined by pooling
the data obtained from each site, and comparisons between groups were
subsequently made. The level of site-specific bone loss was also
determined by separating data by site, and comparisons were made
between groups (2). Overall mean linear (0.204 ± 0.003 mm) and overall area (0.226 ± 0.022 mm2)
measurements were obtained for nonimmunized, unchallenged, age-matched BALB/c mice. Animals orally challenged with P. gingivalis
showed a marked increase in the overall linear measurements compared to
the measurements obtained for the unchallenged group (P < 0.0001) (Table 1). A comparison of
linear and area measurements obtained for each site from unchallenged
and P. gingivalis-challenged mice demonstrated that oral
challenge induced significant bone loss primarily at linear sites 6 and
7 and area site 3 (Tables 1 and 2).
Morphometric analysis of murine maxillary molars indicated that there
was no significant bone loss induced by P. gingivalis oral challenge at linear sites 1 to 5 or area sites 1 and 2 (data not
shown).
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FIG. 3.
Immunoprobe analysis of week 4 murine chamber fluid
samples for P. gingivalis and each of the gingipains. A
10-µl aliquot of P. gingivalis whole cells (~1 × 107 cells) (lanes 1) and 10-µl portions of 1-mg/ml
solutions of RgpA (lane 2) and RgpB (lane 3) reduced in sample buffer
were separated by SDS-PAGE and were transferred to Immobilon P
membranes. The blots were probed with week 4 chamber fluid samples from
nonimmunized animals (A) or from mice immunized with P. gingivalis (B), RgpA (C), or RgpB (D) and developed. The positions
of molecular mass markers are indicated on the left (96, 67, 43, 30, 20.1, and 14.4 kDa from top to bottom).
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TABLE 1.
Linear measurements of maxillary molars obtained from
BALB/c mice 42 days after gavage with P. gingivalis
A7A1-28a
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TABLE 2.
Area measurements of maxillary molars obtained from
BALB/c mice 42 days after gavage with P. gingivalis
A7A1-28a
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Immunization of mice with heat-killed P. gingivalis prior to
oral challenge resulted in significant reductions in both overall and
site-specific bone loss compared to the values obtained for the
P. gingivalis-challenged group (P < 0.0001)
(Table 1), and the results resembled the results obtained for
unchallenged animals (P > 0.9) (Table 1). Likewise,
immunization of mice with RgpA protected against oral bone loss.
Interestingly, the level of protection observed after RgpA immunization
was similar to that observed in animals immunized with heat-killed
P. gingivalis (overall, P > 0.4; linear
site 6, P > 0.8; linear site 7, P > 0.6) (Table 1). In contrast, immunization with RgpB failed to
protect mice from P. gingivalis-induced oral bone loss. The
area measurement data for site 3 confirmed the linear measurement data
(Table 2).
Concluding remarks.
We previously demonstrated that RgpA and
RgpB represent a group of vaccine candidates that could be used for
prevention of P. gingivalis infection (9);
however, it was recognized that the results obtained may not accurately
represent what occurs during P. gingivalis infection in the
oral cavity (11). The results obtained in the present
study demonstrate that immunization of mice with RgpA or heat-killed
P. gingivalis prevents oral bone loss stimulated by P. gingivalis. However, immunization of mice with RgpB failed to
stimulate protection against P. gingivalis oral challenge.
Animals immunized with RgpA possessed modest levels of P. gingivalis-specific IgM and high levels of P. gingivalis-specific IgG, while mice immunized with RgpB had a
modest P. gingivalis-specific IgM response and a P. gingivalis-specific IgG titer significantly lower than that
observed for mice immunized with RgpA. Low levels of P. gingivalis-specific IgM and high levels of P. gingivalis-specific IgG have also been observed following
immunization of rats with P. gingivalis HagB
(16). As RgpA and RgpB possess similar catalytic domains
and only RgpA possesses a hemagglutinin domain, we concluded that
protection and the enhanced P. gingivalis-specific IgG
levels were directed to the hemagglutinin domain of RgpA. This
conclusion was supported by our observation that chamber fluid samples
from RgpA-immunized mice, but not chamber fluid samples from
RgpB-immunized mice, recognized a 44-kDa band in P. gingivalis whole-cell lysates, which corresponded to the major
hemagglutinin domain of RgpA. Similar blots probed with chamber fluid
samples from mice immunized with RgpB failed to detect this band. There
are two possible explanations for this: (i) RgpB is not exposed at high
levels on the surface of P. gingivalis, or (ii) the presence
of the hemagglutinin domain stimulates the production of an antibody
subset that efficiently recognizes P. gingivalis.
O'Brien-Simpson et al. have reported results similar to observations
made in the present study, as immunization of mice with RgpA-Kgp
hemagglutinin domain peptides induced a potent IgG response and
stimulated a protective host response when a murine lesion model was
used (24).
Previously, we reported that immunization of mice with either RgpA or
RgpB stimulated protection against P. gingivalis challenge when the subcutaneous chamber model was used. It is not clear why RgpB
immunization provided protection in the subcutaneous model but failed
to provide protection in the oral challenge model. Based on our
observations, a likely explanation is that immunization with RgpB did
not result in a sufficiently high level of P. gingivalis-specific IgG in serum. As a result, the low levels of
P. gingivalis-specific antibody elicited by RgpB were unable
to modulate either P. gingivalis colonization or subsequent
oral bone loss. It is possible that in the subcutaneous challenge
model, RgpB immunization elicited elevated levels of antigen-primed
immune cells which were localized at this site prior to challenge with
P. gingivalis. This effect could enhance localized P. gingivalis killing and thus suggests that RgpB elicited a
protective response. Booth et al. (4) reported that
localized administration of a P. gingivalis-specific monoclonal antibody at severely infected subgingival sites
significantly reduced subsequent P. gingivalis
recolonization in periodontal patients. Although the protection
observed was transient, this study demonstrated that a specific
antibody is able to promote selective removal of P. gingivalis from the oral cavity. In addition, Klausen et al.
(17) demonstrated that rats immunized with P. gingivalis produced elevated levels of serum and salivary
antibodies and that immunized animals were protected from oral bone
loss elicited by P. gingivalis challenge. Taken together,
these observations, in conjunction with our data, may suggest that a
critical level of P. gingivalis-specific antibody is
necessary to prevent P. gingivalis colonization in the oral cavity.
In summary, we demonstrated that immunization of mice with RgpA can
stimulate production of both P. gingivalis- and
RgpA-specific IgG primarily directed to the hemagglutinin domain of
this protein. The elevated levels of this antibody coincide with
protection against oral bone loss elicited by P. gingivalis.
Taken together, these data suggest that RgpA is a novel vaccine
candidate that could be used for prevention of periodontal disease
caused by P. gingivalis.
We thank Salomon Amar for the use of his light microscopy station
to obtain linear and area measurements and Lee Wetzler for helpful
criticism in preparing the manuscript.
This work was supported in part by Public Health Science grant 12517 awarded to C.A.G. and by National Research Service Award grant
DE05739-02 awarded to F.C.G.
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