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in BALB/c MicePorphyromonas gingivalisRgpA-Kgp Proteinase-Adhesin Complexes of Characterization of T Cell Responses to the

Leanne T. Frazer and Eric C. ReynoldsVivian Tam, Neil M. O'Brien-Simpson, Rishi D. Pathirana,

http://www.jimmunol.org/content/181/6/4150doi: 10.4049/jimmunol.181.6.4150

2008; 181:4150-4158; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/181/6/4150.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Characterization of T Cell Responses to the RgpA-KgpProteinase-Adhesin Complexes of Porphyromonas gingivalis inBALB/c Mice1

Vivian Tam, Neil M. O’Brien-Simpson, Rishi D. Pathirana, Leanne T. Frazer,and Eric C. Reynolds2

Porphyromonas gingivalis is a Gram-negative bacterium strongly associated with chronic periodontitis, an inflammatory oraldisease. A major virulence factor common to all characterized strains of P. gingivalis is the RgpA-Kgp proteinase-adhesin com-plexes (RgpA-Kgp complexes). In this study, we investigated T cell proliferative and cytokine responses to the RgpA-Kgp com-plexes and identified T cell epitopes in BALB/c mice utilizing Pepscan methodology. T cell proliferative responses were found tobe predominantly directed toward the proteinase catalytic domains. Eleven T cell epitopes were identified using RgpA-Kgp-primed lymph node T cells (IL-4 dominant) and 21 using an RgpA-Kgp-specific T cell line (IFN-� dominant), with 5 T cell epitopes,including the immunodominant epitope peptide 22, common to both T cell populations. Peptide 22 (439ANYTAHGSETAWADP453) from the Kgp proteinase catalytic domain induced a Th2 cytokine response in mice, and peptide 22-primed Tcells had a Th2 cytokine profile when stimulated with the RgpA-Kgp complexes. Truncation and alanine scanning of peptide 22identified the minimum epitope (442TAHGSETAWA451), and residues His444, Glu447, and Trp450 as critical for T cell proliferation.With a view to vaccine development, peptide 22 was incorporated into a synthetic peptide polymer. Peptide 22 polymer inducedstrong T cell proliferation and crossreactivity to native RgpA-Kgp complexes. In conclusion, we have identified a major T cellepitope of P. gingivalis and established that antigenicity of the T cell epitope is retained when delivered as a peptide polymer. Thestrategies employed here may have potential in the development of a synthetic peptide vaccine for P. gingivalis. The Journal ofImmunology, 2008, 181: 4150–4158.

C hronic periodontitis is an inflammatory disease associ-ated with specific bacteria that is characterized by thedestruction of the tissues that support the teeth. The dis-

ease is a major health problem, with an annual cost of treatmentestimated at US$14 billion per annum in the United States alone(1). A study by Socransky et al. (2) reported that three bacteria,Tannerella forsythia (previously Bacteroides forsythus), Porphy-romonas gingivalis, and Treponema denticola, are strongly asso-ciated with the severity of chronic periodontitis. Among thesethree bacteria, P. gingivalis has been the most studied and has beenreported to be closely associated with the clinical measures ofperiodontitis, including increased pocket depth and bleeding onprobing, and is commonly recovered from subgingival plaqueof individuals with disease, but is rarely recovered from healthyperiodontal sites (3– 6). P. gingivalis when used to orally chal-lenge mice and rats (7–10) and nonhuman primates (11) inducesperiodontal bone loss. Immunization with killed, whole P. gin-givalis cells protects against P. gingivalis-induced alveolar

bone loss in the mouse (8), rat (9), and in the nonhuman primateperiodontitis models (11), indicating that a vaccine directedagainst P. gingivalis may protect against P. gingivalis-inducedchronic periodontitis.

P. gingivalis is an anaerobic, black pigmented, asacchrolytic,Gram-negative rod which has an absolute requirement for iron,preferably in the form of heme or hemin. The bacterium has anumber of virulence factors, including Arg- and Lys-specific pro-teinases and associated adhesins (termed the RgpA-Kgp com-plexes), fimbriae, LPS, the capsule, outer membrane vesicles,and hemagglutinins (12). These virulence factors have beenfound to vary between P. gingivalis strains (13); however, acommon virulence factor among all characterized P. gingivalisstrains is the RgpA-Kgp complexes (14 –16) (reviewed byO’Brien-Simpson et al. in Ref. 17). The RgpA and Kgp com-plexes are encoded by two genes, rgpA and kgp, respectively.The complexes are associated noncovalently and anchored toLPS on the cell surface of the bacterium (18, 19). The Arg-specific proteinase (RgpA) consists of a 45-kDa Arg-specificcatalytic domain (RgpAcat, formerly RgpA45) and is associatedwith four adhesin domains at the C terminus, namely RgpAA1,RgpAA2, RgpAA2, and RgpAA4 (formerly RgpA44, RgpA15,RgpA17, and RgpA27, respectively) (19, 20). The Lys-specificproteinase (Kgp) consists of a 48-kDa Lys-specific catalytic do-main (Kgpcat, formerly Kgp48), and is associated with a series ofC-terminal adhesin domains, namely KgpA1, KgpA2 (formerlyKgp39 and Kgp15, respectively), KgpA3, KgpA4, and KgpA5 (for-merly known collectively as Kgp44) (19, 21) (Fig. 1). The adhesindomains contain repeated adhesin binding motifs, which are sug-gested to bind to a number of host proteins including fibronectin,fibrinogen, and collage type V (8).

Cooperative Research Centre for Oral Health Science, School of Dental Science, TheUniversity of Melbourne, Victoria, Australia

Received for publication March 19, 2008. Accepted for publication July 14, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Health and Medical Research Council (Aus-tralia) Grant 454475.2 Address correspondence and reprint requests to Dr. Eric C. Reynolds, Centre forOral Health Science, School of Dental Science, Bio21 Molecular Science and Bio-technology Institute, The University of Melbourne, Victoria 3010, Australia. E-mailaddress: e.reynolds@unimelb.edu.au

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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The RgpA-Kgp complexes have been identified as a majorvirulence factor contributing to the pathogenicity of P. gingi-valis, as mutant strains of the bacterium that lack the protein-ases and adhesins are less virulent in the mouse lesion and inperiodontitis models (22, 23). Additionally, when a preparationof the RgpA-Kgp complexes was used as a vaccine, it wasfound to give protection in the mouse lesion and periodontitismodels and in the rat periodontitis model (8, 9, 24). A numberof peptides corresponding to both the proteinases and adhesinswhen conjugated to diphtheria toxoid and used as immunogenswere protective in the mouse lesion and in periodontitis models(8, 24). Studies have also shown that protection in mice is as-sociated with the production of a P. gingivalis Ag-specific IgG1subclass and Th2 IL-4 cytokine response, whereas disease wasassociated with an IgG3 Ab response and a predominant IFN-�(Th1) cytokine response (8, 25).

While the exact role of T cells in periodontitis has yet to befully elucidated, the cells have been reported to be present inthe inflammatory infiltrate during periodontal disease (26). Fur-thermore, studies by Baker et al. (27, 28) have suggested thatCD4� T cells, and not CD8� T cells, are associated with boneresorption after oral infection with P. gingivalis in mice. Otherreports have indicated that the presence of proinflammatory cy-tokines IFN-� and IL-6 produced by CD4� T cells are alsoassociated with bone loss (27) and that IL-10 (a Th2-type cy-tokine) knock-out mice exhibited more P. gingivalis-inducedalveolar bone loss than that of wild-type mice (29). T cells havealso been reported to play a role in protection against bone lossin the mouse periodontitis model, with IL-4 being the predom-inant cytokine secreted by these protective cells (8). Further-more, a bias toward a Th1 response resulted in elevated levelsof periodontal tissue inflammation and alveolar bone loss inmice after challenge with P. gingivalis, whereas mice that werebiased toward a Th2 response did not develop periodontal boneloss (30).

T cells are therefore likely to play an important role in chronicperiodontitis. However, the T cell responses to the RgpA-Kgpcomplexes of P. gingivalis have not yet been determined. In thisstudy, we identify the major T cell stimulatory domains of theRgpA-Kgp complexes, identify the predominant T cell epitopes,and characterize the T cell cytokine responses to the RgpA-Kgpcomplexes. Furthermore, we define the immunodominant epitopeutilizing truncation and alanine scanning techniques and evaluatethe epitope immunogenicity in a peptide polymer vaccineconstruct.

Materials and MethodsGrowth of P. gingivalis and purification of RgpA-Kgp, Kgp,RgpA, and recombinant KgpA1

P. gingivalis strain W50 (31) and P. gingivalis mutant strains RgpA�

(W501) (32) and Kgp� (K1A) (33) were grown as previously described(34). P. gingivalis RgpA-Kgp, RgpA, and Kgp were prepared and charac-terized as previously described (34). Recombinant KgpA1 was produced aspreviously described by Frazer et al. (35).

Immunization protocols

BALB/c mice were obtained from the animal facility at the Department ofImmunology and Microbiology at The University of Melbourne, and ani-mal experimentation was approved by the University of Melbourne animalethics committee. RgpA-Kgp complexes (50 �g/mouse) or peptide 22 (30nmol/mouse) was emulsified in CFA (Sigma-Aldrich) and used to immu-nize BALB/c mice subcutaneously in the hind leg.

T cell proliferation assays

Lymphocytes were prepared from pooled inguinal and popliteal lymphnode cell suspensions of BALB/c mice primed 7 days previously withthe RgpA-Kgp complexes. Spleens were isolated from nonimmunizedmice as a source of syngeneic APCs. Lymph nodes and spleens werecollected in enriched DMEM/Ham’s nutrient mixture F-12 (DMEM/F-12) supplemented with 10% (v/v) heat-inactivated (56°C, 30 min) FBS,2 mM glutamine, 2 mM sodium pyruvate, 0.1 mM 2-ME, 30 �g/mlgentamicin, 100 IU/ml penicillin, and 100 �g/ml streptomycin (JRHBiosciences), L-arginine (116 mg/ml), L-asparagine (36 mg/ml), andfolic acid (6 mg/ml, Sigma-Aldrich), and single-cell suspensions weremade by passing the lymph nodes or spleens through a wire mesh. RBCwere removed from the spleen cell suspension by treatment with am-monium Tris buffer (17 mM Tris-HCl, 140 mM ammonium chloride inMilli-Q water (pH 7.2)) for 5 min at room temperature, and then washedthree times in enriched DMEM/F-12 (5 min, 500 � g, EconoSpin, Sor-vall Instruments, DuPont).

Monocytes and dead cells were removed from the lymph node cell sus-pension using Lympholyte-M (Cedarlane Laboratories) as per the manu-facturer’s instructions. Lymph node T cells were enriched using the mouseT cell recovery columns (Cedarlane Laboratories) as per the manufactur-er’s instructions. T cells were cultured in enriched DMEM/F-12 at a con-centration of 3 � 105 lymph node T cells/well in a 96-well microtiter plate(Nunc) in the presence of syngeneic �-irradiated (2200 rads) spleen cells(3 � 105cells/well) together with Ag (RgpA-Kgp, RgpA, and Kgp, recom-binant KgpA1 or peptide) in a total volume of 250 �l. T cells were incu-bated for four days at 37°C in an atmosphere of 5% CO2 in air. One �Ci[3H]thymidine (Amersham Biosciences) was added per well and incubatedfor a further 18 h. Cells were then lysed with cell lysis buffer (30 mMTris-HCl, 100 mM EDTA, 1% N-lauroylsarcosine (pH 8)) and harvestedonto glass-fiber filters using a Tomtec Harvester96 Mach III cell harvester.The glass-fiber filters were then air dried and sealed in plastic bags con-taining 5 ml of Betaplate Scint (PerkinElmer), and incorporation of[3H]thymidine was measured using a Wallac MicroBeta Trilux liquid scin-tillation counter (PerkinElmer). Data are expressed as stimulatory index(S.I.)3 � SD, where S.I. is the cpm divided by the negative control (noAg) cpm.

Generation of an RgpA-Kgp-specific T cell line

Lymphocytes were prepared from pooled inguinal and popliteal lymphnode cell suspensions from BALB/c mice primed 7 days previously withthe RgpA-Kgp complexes and spleens were isolated from nonimmunizedmice as a source of syngeneic APCs. Isolated T cells were resuspended inenriched DMEM/F-12 at a concentration of 2 � 105 T cells/ml and incu-bated with syngeneic �-radiated (2200 rads) spleen cells (2 � 106 cells/ml),and 1 �g/ml RgpA-Kgp Ag for 5day s. The T cells were subsequentlyexpanded using 25 U/ml of human recombinant IL-2 (rhIL-2, Sigma-Aldrich) in enriched DMEM/F-12 for a further 5 days before being isolatedwith Lympholyte-M. The cycle of Ag stimulation and rhIL-2 expansionwas then repeated to maintain the RgpA-Kgp-specific T cell line.

ELISPOT assay

ELISPOT assay was performed as previously described (8) using theRgpA-Kgp complexes or peptide 22 as the stimulating Ag.

3 Abbreviations used in this paper: S.I., stimulatory index; rhIL-2, recombinant hu-man IL-2.

FIGURE 1. Schematic representation of RgpA and Kgp polyproteinsshowing processing into catalytic (cat) and adhesin (A) domains.

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Peptide synthesis and purification

Unless otherwise stated, chemicals were of peptide synthesis grade or itsequivalent. O-benzotriazole-N,N,N�,N�-tetramethyluronium hexafluoro-phosphate (HOBt), diisopropylethylamine (DIPEA), N,N-dimethylfor-mamide (DMF), piperidine, trifluoroacetic acid (TFA), and 9-fluorenyl-methoxycarbonyl (Fmoc) protected amino acids were obtained fromAuspep.

Overlapping, 15-mer peptides (offset by 10 residues and overlappingby 5 residues) representing the sequence of the RgpA-Kgp from P.gingivalis (strain W50) were manually synthesized on diketopiperazinepins (Mimotopes) using standard solid-phase peptide synthesis proto-cols for Fmoc chemistry. The Fmoc group was removed with 20% (v/v)piperidine in DMF (20 min). After washing (DMF 2 min, methanol 4 �2 min), the pins were air dried (40 min). Acylation was accomplishedwith HBTU ((O-(1H-benzotriazole-1-yl)-N,N,N�,N�-tetramethyluro-nium hexafluorophosphate))/HOBt activation with 4 equivalents ofFmoc-amino acid and 6 equivalents of DIPEA with respect to pin sub-stitution for 4 h, and was followed by a further acylation (double cou-pling) for 18 h. Before cleavage from the pins, the side-chain protectinggroups were removed by submerging the pins in TFA/ethanedithiol/anisole (95:2.5:2.5) for 2.5 h at room temperature with gentle agitation.After removal of the side-chain protecting groups, the pins werewashed: methanol (10 min), 0.5% (v/v) acetic acid in methanol/water(1:1 (v/v), 1 h), and then Milli-Q water (5 min). Cleavage of the pep-tides from the pins was achieved by submerging the pins in 0.1 Mammonium bicarbonate (pH 8.4) containing 40% (v/v) acetonitrile for30 min with sonication. Cleaved peptides were lyophilized and storedat �20°C.

Peptide 22 (439ANYTAHGSETAWADP453) was synthesized on Fmoc-PAL-PEG-PS resin (PerSeptive Biosystems) using an AB 431A peptidesynthesizer (Applied Biosystems) and standard solid-phase synthesis pro-tocols for Fmoc chemistry.

Acryloyl-amino hexanoic acid (Ahx) peptide 22 used for polymerizationwas synthesized and purified by reversed-phase HPLC as previously de-scribed (8, 36). The purified peptides were analyzed by mass spectrometry,using a Voyager DE MALDI-TOF mass spectrometer (PerSeptive Biosys-tems) and had the observed masses of 1589.76 Da for peptide 22 (calcu-lated mass 1589.65 Da), and 1757.23 Da for acryloyl-Ahx-peptide 22 (cal-culated mass of 1756.86 Da).

Polymerization of acryloyl peptides

Polymerization of acryloyl peptide 22 monomer (acryloyl-Ahx-peptide22) was achieved by the addition of 50-fold molar excess of acrylamide(with respect to the amount of acryloylated peptide) and was initiatedby the addition of ammonium persulfate and N,N,N�,N�-tetramethyleth-ylenediamine (TEMED). The reaction was conducted in 6 M guanidine-HCl containing 2 mM EDTA and 0.5 M Tris (pH 8.3) and was left atroom temperature for 18 h under nitrogen. Polymer was purified using

size-exclusion chromatography on a Superose 12 column 10/300 GL(10 � 300 mm) installed on a Waters Delta 600/Millenium HPLC sys-tem (Waters). The chromatogram was developed at a flow rate of 0.5ml/min using 50 mM ammonium bicarbonate and monitored at 280 nm.Material eluted in the void volume from the column was collected.

Statistical analysis

The alanine scan data were found to be not normally distributed usingLevene’s test of homogeneity of variances (SPSS for Windows, release6.0; SPSS); hence, the S.I. for the alanine scan was statistically ana-lyzed using the Kruskal-Wallis test and the Mann-Whitney U Wilcoxonrank sum test with a Bonferroni correction for type 1 error (SPSS forWindows, release 6.0; SPSS) (37). The S.I. for the epitope mapping and

FIGURE 2. Stimulation of RgpA-Kgp-primed lymph node T cells withRgpA-Kgp (�), RgpA (f), Kgp (Œ), and recombinant KgpA1 (�). De-tection of T cell proliferation was measured using [3H]thymidine incorpo-ration and compared with nonstimulated T cells (‚). Data are expressed asthe S.I. and are the average of triplicate assays � SD.

FIGURE 3. Stimulation of RgpA-Kgp-primed BALB/c mouse lymphnode T cells (f) and RgpA-Kgp-specific BALB/c mouse T cell line (�)with RgpA-Kgp complexes (A). Detection of proliferation was mea-sured using [3H]thymidine incorporation and is expressed as the S.I.Means are the average of triplicate assays � SD. Cytokine profile ofRgpA-Kgp complexes stimulated RgpA-Kgp-primed BALB/c lymphnode T cells (d � 21.87, 99.9%, CI of 0.56, 11.59) (B) and RgpA-Kgp-primed BALB/c T cell line cells (d � �11.13, 99.9%, CI of �24.17,0.33) (C). Lymph node T cells and T cell line T cells were stimulatedwith 3.125 and 10 �g/ml of RgpA-Kgp complexes for 48 h, respec-tively. Results are expressed as spot-forming cells per million and arethe average of triplicate assays � SD.

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the ELISPOT data were analyzed for significance utilizing a one-wayclassification ANOVA with a posthoc Dunnett’s T3 test. Effect sizes,represented as Cohen’s (38) d, were calculated using the effect sizecalculator provided online by Evidence-Based Education U.K. web site(http://cem.dur.ac.uk/ebeuk/researc/effectsize/). According to Cohen(38), a small effect size is d of �0.2 and �0.5, a moderate effect sizeis d of �0.5 and �0.8, and a large effect size is d of �0.8.

ResultsT cell responses to RgpA-Kgp, RgpA, and Kgp

The antigenicity of the different components of the RgpA-Kgpcomplexes were investigated by stimulating RgpA-Kgp-primed Tcells with a range of concentrations of RgpA-Kgp, RgpA, Kgp,and recombinant KgpA1 adhesin (rKgpA1) (Fig. 2). All the Ags thatwere tested stimulated T cell proliferation of RgpA-Kgp-primed Tcells, but each Ag induced maximum T cell proliferation at dif-ferent Ag concentrations. The Kgp Ag induced maximum prolif-eration at an Ag concentration of 0.78 pmol/ml, whereas the RgpAand RgpA-Kgp Ags induced maximum proliferation at concentra-tions of 1.56 and 6.25 pmol/ml, respectively. Recombinant KgpA1

induced maximum proliferation at the highest antigenic concen-tration of 200 pmol/ml. At an antigenic concentration of 0.78pmol/ml, there was no significant difference in the proliferation ofthe RgpA-Kgp-primed T cells in response to the Kgp, RgpA, andRgpA-Kgp complexes; however, at that concentration, rKgpA1 in-duced significantly ( p � 0.05) lower levels of T cell proliferationthan did the proteinase Ags. The RgpA-Kgp complexes induced ahigher maximum response than that of either RgpA or Kgp, andthere was no significant difference in the maximum response in-duced by RgpA or Kgp.

Cytokine and proliferative responses of RgpA-Kgp-primedlymph node T cells and a RgpA-Kgp-specific T cell line

To establish an RgpA-Kgp-specific T cell line, lymph node T cellspreviously primed with the RgpA-Kgp complexes were culturedwith the RgpA-Kgp complexes for 5 days and then expandedwith rhIL-2 for a further 5 days. This cycle was repeated to es-tablish a RgpA-Kgp-specific T cell line. The proliferative andcytokine responses induced by the RgpA-Kgp-specific T cellline and RgpA-Kgp-primed lymph node T cells were evaluatedand compared (Fig. 3). The proliferative response of the T cellline when stimulated with the RgpA-Kgp Ag at 25 pmol/ml wastwice as strong as the proliferative response of the RgpA-Kgp-primed lymph node T cells. The maximum T cell proliferationwas observed at similar Ag concentrations (25 pmol/ml) forboth cell types (Fig. 3A).

The cytokine secretion profile of the RgpA-Kgp-primed lymphnode T cells and the RgpA-Kgp-specific T cell line is shown inFig. 3, B and C. Stimulation of the lymph node T cells with theRgpA-Kgp complexes induced a predominant IL-4 cytokine re-sponse (Fig. 3B), where the number of cells producing IL-4 wassignificantly higher ( p � 0.05) than the number of cells producingIFN-�. Stimulation of the RgpA-Kgp-specific T cell line induceda predominant IFN-� response (Fig. 3C), where the number ofcells producing IFN-� was significantly higher ( p � 0.01) than thenumber of cells producing the IL-4 cytokine.

0 1 2 3 4 5 6 7

0 1 2 3 4 5 6 71-56-1011 -1516-2021-2529-3031-3536-4041-4546-5051-5556-6061-6566-7071-7576-8081-8586-9091-9596-100141-145146-150151-155156-160161-165166-170171-175176-180

PeptideGroups

Kgpcat

KgpA1

RgpAcat

KgpA2

B

RgpAcat

161718192021222324253637383940156157158159160

Peptides

Kgpcat

C

T-cell proliferative responseStimulatory Index (S.I. Units)

12

KgpA3, A4, A5

0 2 4 6 8 101-2021-4041-6061-8081-100101-120121-140141-160161-180181-192

PeptideGroups Kgpcat

KgpA1KgpA2

RgpAcat

RgpAA1

A Domains ofthe RgpAand Kgp

Peptides

0 1 2 3 4 5 6 7 81112131415161718191101111121131141151161171181191

D

Kgpcat

KgpA1

KgpA2

RgpAcat

RgpAA1

KgpA3, A4, A5

FIGURE 4. RgpA-Kgp-primed lymph node T cells stimulated with syn-thetic peptides. The 192 15-mer overlapping peptides representing theunique sequences of RgpA and Kgp were pooled into groups of 20 peptidesand used to stimulate RgpA-Kgp-primed lymph node T cells (A). Thegroups of 20 peptides that were found to induce T cell proliferation werefurther divided into groups of 5 peptides and used to stimulate RgpA-Kgp-primed lymph node T cells (B). The groups of 5 peptides that were foundto induce T cell stimulation of �2 S.I. units were further divided into single

peptides and used to stimulate RgpA-Kgp-primed lymph node T cells (C).RgpA-Kgp-specific BALB/c mouse T cell line cells stimulated with 192individual overlapping peptides representing the unique sequences ofRgpA and Kgp (D). Proliferation was measured using [3H]thymidine in-corporation and is expressed as the S.I. Means are the average of triplicateassays � SD.

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Identification of RgpA-Kgp T cell epitopes

As the cytokine profiles of the RgpA-Kgp-primed lymph node Tcells and the RgpA-Kgp-specific T cell line were different, both Tcell populations were used to identify RgpA-Kgp T cell epitopes.Pepscan methodology was used to identify the T cell epitopes. Theadhesin domains of RgpA and Kgp share a high degree of aminoacid sequence identity, and thus to gain full sequence coverage ofthe RgpA-Kgp complexes, a series of 192 overlapping 15-mer pep-tides representing the sequences of RgpA proteinase and its ad-hesins, the Kgp proteinase domain and the unique Kgp adhesinsequences, were synthesized using standard Fmoc chemistry.These overlapping 15-mer peptides represented full amino acidsequence coverage of both RgpA and Kgp. Initially, peptides werepooled into groups of 20 peptides to identify immunodominantregions and to facilitate rapid identification of T cell epitopes usingAg-primed lymph node T cells. The T cell proliferative response ofthe RgpA-Kgp-primed T cells to each of the groups of pooled 20peptides is shown in Fig. 4A. Peptide pools corresponding toKgpcat, KgpA1, KgpA2, and RgpAcat stimulated proliferation ofRgpA-Kgp-primed lymph node T cells. Peptides corresponding toKgpcat (group 21–40) stimulated the strongest proliferation, fol-lowed by peptides corresponding to RgpAcat (group 161–180),whereas peptides corresponding to KgpA1 (groups 41–60 and 61–80) and KgpA2 (group 81–100) stimulated a weaker T cell re-sponse. The groups of 20 peptides that stimulated proliferation ofthe RgpA-Kgp-primed lymph node T cells were then divided intogroups of 5 peptides and used to stimulate the same type of cells(Fig. 4B). Only peptide groups corresponding to Kgpcat (peptidegroups 16–20, 21–25, and 36–40) and RgpAcat (peptide group156–160) stimulated a T cell proliferative response. The individ-ual peptides from each of these groups were then used to stimulateRgpA-Kgp-primed lymph node T cells (Fig. 4C) to identify themajor immunodominant peptides. Eleven peptides were found toinduce proliferation of the RgpA-Kgp-primed lymph node T cells(Fig. 4C). Peptide 22 was found to induce the strongest T cellproliferation, followed by peptides 158 and 23. Peptides 16, 17, 18,20, 37, 39, and 40 (from Kgpcat) stimulated weaker T cell prolif-eration in comparison to peptides 22, 23, and 158. The sequences

of the identified peptides containing T cell epitopes from thelymph node T cells are summarized in Table I.

The 192 individual peptides were also used to stimulate theRgpA-Kgp-specific T cell line. Twenty-one RgpA-Kgp T cellepitopes were identified (Fig. 4D). Seventeen of these epitopeswere located within Kgpcat, two epitopes were located withinKgpA1, and one epitope was located in each of the KgpA2/RgpAA2

and RgpAcat domains. The sequences of these epitopes are shownin Table I. Peptides 15, 17, 22, 39, and 43 located in Kgpcat domainstimulated the strongest T cell proliferative response in the RgpA-Kgp-specific T cell line. Five of the identified epitopes (peptides22, 23, 39, 16, and 17) were identified in both the RgpA-Kgp-primed lymph node T cells and RgpA-Kgp-specific T cell line(Table I). Peptide 22 (ANYTAHGSETAWADP) was found to in-duce a consistently stronger T cell proliferative response comparedwith the other epitopes.

Characterization of the T cell responses induced by the Kgpcat

sequence ANYTAHGSETAWADP (peptide 22)

Peptide 22 was synthesized using standard solid-phase synthesisprotocols for Fmoc chemistry, purified using reversed-phaseHPLC purification and subsequently utilized in T cell prolifer-ation and ELISPOT assays. Peptide 22 induced maximum T cellproliferation at 25 nmol/ml for both RgpA-Kgp-primed T cellsand peptide 22-primed T cells, but stimulated a higher prolif-erative response from the peptide 22-primed T cells (Fig. 5, Aand B). Peptide 22-primed T cells were tested with the RgpA-Kgp Ag to evaluate the ability of peptide 22-primed cells to bestimulated by native Ag (Fig. 5C). Maximum proliferation in-duced by the RgpA-Kgp complexes in peptide 22-primed lymphnode T cells was at an Ag concentration of 12.5 pmol/ml, whichwas approximately 4-fold higher than the RgpA-Kgp concen-tration required to maximally stimulate RgpA-Kgp-primed Tcells (Fig. 5C).

The cytokine secretion profiles of the peptide 22-primed andRgpA-Kgp-primed T cells when stimulated with peptide 22 werealso investigated (Fig. 6). RgpA-Kgp-primed T cells had a higher

Table I. Sequences of the peptides that stimulated proliferation of RgpA-Kgp-specific T cells

Lymph Node T Cells T Cell Line Cells

Peptide No. Peptide Sequence Origin S.I. Units Peptide No. Peptide Sequence Origin S.I. Units

16a 379EKVLLIAGADYSWNS393 Kgpcat 2.49 7 289AFIHKKYNDGLAASA303 Kgpcat 2.0617a 389YSWNSQVGQPTIKYG403 Kgpcat 2.72 10 319GEKGKKTKKVTDLYY333 Kgpcat 3.5618 399TIKYGMQYYYNQEHG413 Kgpcat 2.31 15 369KATMPDKSYLEKVLL383 Kgpcat 7.3620 419NYLKAPYTGCYSHLN433 Kgpcat 2.14 16a 379EKVLLIAGADYSWNS393 Kgpcat 2.0522a 439ANYTAHGSETAWADP453 Kgpcat 6.66 17a 389YSWNSQVGQPTIKYG403 Kgpcat 2.0423a 449AWADPLLTTSQLKAL463 Kgpcat 3.19 21 429YSHLNTGVSFANYTA443 Kgpcat 2.4737 589GDGSVMPYRAMPKTN603 Kgpcat 2.09 22a 439ANYTAHGSETAWADP453 Kgpcat 3.5739a 609ASLPQNQASYSIQAS623 Kgpcat 2.68 23a 449AWADPLLTTSQLKAL463 Kgpcat 2.1540 619SIQASAGSYVAISKD633 Kgpcat 2.71 27 489FGEVITRVKEKGAYA503 Kgpcat 3.91158 231TNSNQLPFIFDVACV245 RgpAcat 3.23 28 499KGAYAYIGSSPNSYW513 Kgpcat 2.15159 241DVACVNGDFLFSMPC255 RgpAcat 2.01 34 559ANGLAATHAGNIGNI573 Kgpcat 3.6

39a 609ASLPQNQASYSIQAS623 Kgpcat 5.8941 629AISKDGVLYGTGVAN643 Kgpcat 3.5843 649TVSMTKQITENGNYD663 Kgpcat 4.0445 669SNYLPVIKQIQVGEP683 Kgpcat 2.4446 709SAKKAEGSREVKRIG723 Kgpcat 2.9447 719VKRIGDGLFVTIEPA733 Kgpcat 3.0751 758QFLLDADHNTFGSVI772 KgpA1 2.6967 918EYCVEVKYTAGVSPK932 KgpA1 2.1191 1157ADFTETFESSTHGEA1171 KgpA2 2.21162 271GTVAIIASTINQSWA285 RgpAcat 2.49

a Epitopes that were identified from both the RgpA-Kgp-primed lymph node T cells and the RgpA-Kgp-specific T cell line.

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IL-4 response in comparison with IFN-� secretion when stimu-lated with RgpA-Kgp complexes ( p � 0.05, average effect sized � 21.87) or peptide 22 (average effect size d � 4.33) (Fig. 6).Peptide 22-primed T cells had a higher IL-4 response when stim-ulated with RgpA-Kgp complexes (average effect size d � 3.82);however, the differences in IL-4 and IFN-� secretion induced bypeptide 22 (average effect size d � 0.67) were not significant(Fig. 6).

Identification of the critical residues and minimal T cellepitope of peptide 22 (ANYTAHGSETAWADP)

To identify the minimum T cell epitope of peptide 22, 15 15-merpeptides overlapping by 14 residues and offset by 1 residue fromresidues Ala432 to Leu460 (Table IIA) were used to stimulate pep-tide 22-primed lymph node T cells (Fig. 7A). The minimumepitope for peptide 22 was the inner sequence TAHGSETAWA,where the removal of the N-terminal Thr and C-terminal Ala bothresulted in no T cell proliferation of the peptide 22-primed T cells.Peptide 9 (NYTAHGSETAWADPL), which has the N-terminalAla of peptide 22 deleted, was found to induce a significantly ( p �

0.05) stronger T cell proliferative response than did the originalsequence.

The alanine scanning technique was used to investigate whichresidues in peptide 22 are important for T cell proliferation (Fig.7B). Twelve peptides (Table IIB) were used to stimulate peptide22-primed T cells and the proliferative responses were compared

FIGURE 5. Peptide 22 stimulation of RgpA-Kgp-primed lymph nodeT cells (�) (A) and peptide 22-primed lymph node T cells (f) (B) (Erepresents unstimulated cells). RgpA-Kgp Ag stimulation of RgpA-Kgp-primed lymph node T cells (�) and peptide 22-primed lymph nodeT cells (f) (C). Proliferation was detected using [3H]thymidine incor-poration and is expressed as the S.I. Means are the average of triplicateassays � SD.

FIGURE 6. Cytokine profile of RgpA-Kgp-primed or peptide 22-primed lymph node T cells stimulated with RgpA-Kgp complexes or pep-tide 22. Lymph node T cells were stimulated with 10 �g/ml of RgpA-Kgpcomplex or 50 nmol/ml peptide 22 for 48 h; IL-4 stimulation is shown bythe open bars and IFN-� stimulation is shown by the filled bars. Results areexpressed as spot-forming cells per million and are the average of triplicateassays � SD.

Table II. Sequences of the peptides used in the identification of theminimum epitope (A) and alanine screen (B) of peptide 22

A B

PeptideNo. Peptide Sequence

PeptideNo. Peptide Sequence

1 LNTGVSFANYTAHGS 47 ANYTAHGSETAWADP2 NTGVSFANYTAHGSE 48 AAYTAHGSETAWADP3 TGVSFANYTAHGSET 49 ANATAHGSETAWADP4 GVSFANYTAHGSETA 50 ANYAAHGSETAWADP5 VSFANYTAHGSETAW 51 ANYTAAGSETAWADP6 SFANYTAHGSETAWA 52 ANYTAHASETAWADP7 FANYTAHGSETAWAD 53 ANYTAHGAETAWADP8 ANYTAHGSETAWADP 54 ANYTAHGSATAWADP9 NYTAHGSETAWADPL 55 ANYTAHGSEAAWADP10 YTAHGSETAWADPLL 56 ANYTAHGSETAWADP11 TAHGSETAWADPLLT 57 ANYTAHGSETAAADP12 AHGSETAWADPLLTT 58 ANYTAHGSETAWADP13 HGSETAWADPLLTTS 59 ANYTAHGSETAWAAP14 GSETAWADPLLTTSQ 60 ANYTAHGSETAWADA15 SETAWADPLLTTSQL

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(Fig. 7B). The substitution of the His444, Glu447, and Trp450 resi-dues with Ala resulted in no T cell proliferation of the peptide22-primed T cells. Substitution of Thr442 and Gly445 and Pro453

residues with Ala all had a similar effect at significantly ( p � 0.05)reducing T cell proliferation (average effect size d � �2.77).

Characterization of the T cell proliferative response inducedby peptide 22 polymer and crossreactivity of the peptidepolymer to the native Ag

A peptide polymer strategy (36) that may have utility in theproduction of a multivalent peptide vaccine is the acryloylationand subsequent polymerization of synthetic peptides. To assessthis strategy with P. gingivalis epitopes, peptide 22 was acry-loylated and polymerized, and the ability of the peptide 22 poly-mer to induce a T cell response was evaluated and comparedwith that of peptide 22 monomer (Fig. 8A). Lymph node T cellsthat were primed with peptide 22 were stimulated with bothpeptide 22 and peptide 22 polymer (Fig. 8A). Both peptide 22monomer and peptide 22 polymer induced significant T cellproliferation. Maximum proliferation for both peptide 22 mono-mer and peptide 22 polymer was induced at 12.5 nmol/ml ofAg. The peptide 22 polymer was also tested for its crossreac-tivity with the native RgpA-Kgp complexes. Peptide 22 poly-mer-primed lymph node T cells were stimulated with the RgpA-Kgp complexes and the resultant proliferation is shown in Fig.8B. The RgpA-Kgp complexes stimulated peptide 22 polymer-primed T cells with a maximum proliferation at 50 pmol/ml ofthe RgpA-Kgp complexes.

DiscussionIn this study, we investigated the T cell response and identified Tcell epitopes to different components of the RgpA-Kgp complexesusing RgpA-Kgp-primed T cells and a RgpA-Kgp-specific T cellline. The RgpA-Kgp Ag induced a strong T cell proliferative re-sponse and a predominant IL-4 cytokine response from the RgpA-Kgp-primed lymph node T cells. Using these cells, the abilities ofthe different components of the RgpA-Kgp complexes were eval-uated and the responses compared.

The RgpA-Kgp complexes, as well as the major componentsRgpA and Kgp, induced significant T cell proliferation at low Agconcentrations. However, stimulation of the RgpA-Kgp-primed Tcells with high concentrations of RgpA, Kgp, and RgpA-Kgp re-sulted in reduced levels of T cell proliferation. This phenomenonmay be explained by overstimulation with Ag leading to apoptosis,which has been reported in previous studies (39), but this effectmay also be enhanced by the RgpA and Kgp proteinases, as theseenzymes at high concentrations have been reported to induce ap-optosis in other cell types (40–44). The substantially higher max-imum T cell proliferative response induced by Kgp, RgpA-Kgp,and RgpA at low Ag concentrations compared with the adhesindomain rKgpA1 is consistent with the immunodominant T cellepitopes being localized in the proteinase catalytic domains.

To aid in T cell epitope identification, an RgpA-Kgp-specific Tcell line was generated and was found to produce significantlyhigher levels of IFN-� in comparison with IL-4 after stimulation

FIGURE 7. A, Minimum epitope identification of peptide 22 utilizingpeptide 22-primed lymph node T cells († denotes the native peptideANYTAHGSETAWADP). B, Alanine scan of peptide 22 utilizing pep-tide 22-primed lymph node T cells. Proliferation was measured using[3H]thymidine incorporation and is expressed as the S.I. Results are theaverage of triplicate assays � SD. �, Significantly different at p � 0.05.

FIGURE 8. T cell proliferation induced by peptide 22 polymer. A, Stim-ulation of peptide 22-primed lymph node T cells with peptide 22 (�) andpeptide polymer 22 (f). B, Stimulation of peptide 22 polymer-primed Tcells with RgpA-Kgp complexes (�). Proliferation was measured using[3H]thymidine incorporation and is expressed as the S.I. Data are the av-erage of triplicate assays � SD.

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with the RgpA-Kgp complexes, which was in contrast to that pro-duced by RgpA-Kgp-primed lymph node T cells. The strongIFN-� response induced by the T cell line indicates that the T cellline is of a Th1 cytokine-secreting phenotype. This phenotype maybe attributable to the continual stimulation with the RgpA-Kgpcomplexes, which contain immunogenic LPS-like carbohydrates(45) that have been reported to be crossreactive with P. gingivalisLPS (19, 46). Previous work has reported that stimulation of Tcells with LPS results in the production of local inflammation andTh1 immunoreactivity, including the production of a predominantIFN-� cytokine response (47, 48).

T cell epitopes of the RgpA-Kgp complexes were identified uti-lizing RgpA-Kgp-primed lymph node T cells and the RgpA-Kgp-specific T cell line. Eleven T cell epitopes were identified usinglymph node T cells and 21 T cell epitopes were identified using theT cell line. Five common T cell epitopes of the RgpA-Kgp-primedlymph node T cells and the RgpA-Kgp complex T cell line wereidentified. The Th1 cytokine phenotype nature of the RgpA-Kgp-specific T cell line shown by the predominance of IFN-� produc-tion may have resulted in the different epitopes that were identi-fied. Previous studies using wild-type mice and mice geneticallydeficient in IL-4 (IL-4�/�) or IL-12 (IL-12�/�) have reported thatTh1 and Th2 cells can recognize unique epitopes, as well as somecommon T cell epitopes (49). Taken together, this suggests thatmost T cell epitopes identified using the RgpA-Kgp-specific T cellline may be Th1 T cell epitopes.

A considerably higher proportion of T cell epitopes were iden-tified from the proteinase catalytic domains, with 81% of theepitopes having been identified from the Kgpcat proteinase domainalone. The strong T cell epitopes to the RgpA-Kgp-primed lymphnode T cells appear to be localized around the region of the activesites of the Kgpcat and RgpAcat proteinases, where the two stron-gest peptides (peptides 22 and 158) contain the catalytic histidineand cysteine residues, respectively, that form part of the proteinaseactive site of the Kgp and RgpA/B proteinases, respectively (50).These data indicate that the T cell proliferative responses to theRgpA-Kgp complexes are primarily associated with theproteinases.

The initial Pepscan screening identified peptide 22 as the im-munodominant peptide; however, further analysis revealed that themost effective epitope at inducing T cell proliferation was peptide22 minus the N-terminal alanine (NYTAHGSETAWADP). Thisindicates that the identification of the actual immunodominant Tcell epitope within a given sequence requires at least two Pepscanstudies: the initial Pepscan for the identification of the T cell stim-ulatory sequence, and the latter to define the immunodominant Tcell epitope. The immunodominant T cell epitope was further de-fined by identifying the critical residues important for T cell stim-ulation by replacing each residue with alanine and analyzing theirability to induce T cell proliferation. In these experiments, threeresidues (His444, Glu447, and Trp450) were found to be critical forT cell proliferation, and three residues (Thr442, Gly445, and Pro453)when changed to Ala significantly reduced T cell proliferation.

A number of studies have reported that a protective immuneresponse against P. gingivalis-induced lesions and bone loss ischaracterized by a specific antiinflammatory Ab isotype responseand that this is associated with a P. gingivalis-specific predominantTh2 (IL-4) cytokine response (8, 9, 25). In contrast, P. gingivalis-induced bone loss in animal models and chronic periodontitis inhumans have been characterized by a proinflammatory Ab re-sponse and a Th1 (IFN-�) cytokine response (8, 25, 30). For aneffective vaccine, it is important that the vaccine induces the de-sired Th cytokine response for protective immunity, and that thisis perpetuated by the native Ag. Peptide 22 was found to induce a

Th2 (IL-4) cytokine response, but, more importantly, peptide 22-primed T cells were able to be stimulated by native Ag to producea Th2 (IL-4) response. We have previously identified and charac-terized PAS1K as a protective B cell epitope against P. gingivalis-induced lesion formation and periodontal bone loss when the pep-tide was conjugated with diphtheria toxoid and used as a vaccinein mice (8, 24). PAS1K is a 21-residue peptide that contains thepeptide 22 sequence. PAS1K when conjugated to diphtheria toxoidwas as effective at providing protection as the native RgpA-KgpAg against P. gingivalis (8, 24). Thus, the PAS1K sequence con-tains both Ag-specific B cell and T cell epitopes, and with theadditional T cell help provided by diphtheria toxoid, may explainwhy this peptide was so effective at inducing protection.

Although peptides can be straightforwardly synthesized and pu-rified, a number of problems arise when peptides alone are used asimmunogens. Synthetic peptides alone are small and poorly im-munogenic, and thus they require adjuvants or conjugation to alarger carrier protein. However, with peptide-protein conjugates,there is the problem of carrier-induced epitope-specific suppres-sion, which reduces the Ab response to the peptide of interest (51).A way in which to circumvent the problems associated with pep-tides is by increasing the size and copy number of peptides usinga peptide polymer strategy (36). Using that strategy, the peptide 22polymer prepared induced a T cell proliferative response that wascrossreactive with the native RgpA-Kgp Ag. While the T cell re-sponse to the peptide polymer was weaker than to the peptidemonomer, the immunogenicity of polymer could possibly be en-hanced by the incorporation of a spacer between the peptide andpolymer backbone. The use of other polymer strategies such aspoly(L-lactic acid) (PLA) and poly(D,L-lactic/glycolic acid)(PLGA) for the incorporation of peptides and proteins has beenreported to successfully enhance the immunogenicity of peptideAgs and to induce the production of Ag-specific Abs (52–54). Themultivalent polymer strategy is therefore an effective way for theincorporation of epitopes into a single synthetic construct that stillretains peptide antigenicity, where incorporation of multipleepitopes into polymers may potentially overcome the major hurdleof MHC restriction.

In conclusion, the T cell responses to the RgpA-Kgp complexeswere predominantly directed to the proteinase catalytic domains.Furthermore, an immunodominant epitope was located within theKgp proteinase catalytic domain and induced a Th2 cytokine re-sponse. The epitope as a synthetic peptide was incorporated into apolymer as an effective immunogen. This polymer approach,where multiple epitopes could be incorporated into a multivalentconstruct, may have potential in the development of a syntheticvaccine against P. gingivalis.

AcknowledgmentsWe thank Professor Stephen Kent and Dr. Rob de Rose (The Departmentof Microbiology, The University of Melbourne, Australia) for the use ofthe AID ELISPOT plate reader.

DisclosuresThe authors have no financial conflicts of interest.

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