INFECTION AND IMMUNITY,0019-9567/00/$04.0010

Feb. 2000, p. 732–739 Vol. 68, No. 2

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Expression and Immunogenicity of Hemagglutinin A fromPorphyromonas gingivalis in an Avirulent Salmonella enterica

Serovar Typhimurium Vaccine StrainEMIL KOZAROV,1* NAOHISA MIYASHITA,2† JACOB BURKS,1 KAREN CERVENY,1

THOMAS A. BROWN,1 WILLIAM P. MCARTHUR,1 AND ANN PROGULSKE-FOX1

Department of Oral Biology and the Periodontal Disease Research Center, University of Florida,Gainesville, Florida 32610,1 and Research Center for Advanced Science and Technology,

University of Tokyo, Komaba 4-6-1, Meguroku, Tokyo, Japan2

Received 23 July 1999/Returned for modification 2 September 1999/Accepted 2 November 1999

Porphyromonas gingivalis is a major etiologic agent of periodontitis, a chronic inflammatory disease thatultimately results in the loss of the supporting tissues of the teeth. Previous work has demonstrated theusefulness of avirulent Salmonella enterica serovar Typhimurium strains as antigen delivery systems forprotective antigens of pathogens that colonize or cross mucosal surfaces. In this study, we constructed andcharacterized a recombinant S. enterica serovar Typhimurium avirulent vaccine strain which expresses hem-agglutinin A and carries no antibiotic resistance markers. HagA, a major virulence-associated surface protein,is a potentially useful immunogen that contains an antigenic epitope which, in humans, elicits an immuneresponse that is protective against subsequent colonization by P. gingivalis. The hagA gene, including itspromoter, was cloned into a balanced-lethal Salmonella vector and transferred to the vaccine strain. Heterol-ogous expression of HagA was demonstrated in both Escherichia coli JM109 and S. enterica serovar Typhi-murium vaccine strain x4072. The HagA epitope was present in its native configuration as determined byimmunochemistry and immunoelectron microscopy. Purified recombinant HagA was recognized by sera frommice immunized with the S. enterica serovar Typhimurium vaccine strain. The HagA-specific antigen of thevaccine was also found to be recognized by serum from a periodontal patient. This vaccine strain, whichexpresses the functional hemagglutinin protein, induces a humoral immune response against HagA and maybe useful for developing a protective vaccine against periodontal diseases associated with P. gingivalis.

Porphyromonas gingivalis is considered a major etiologicagent of adult and refractory periodontal disease. Hemagglu-tinins are bacterial surface proteins that often function as ad-hesins by which bacteria attach to host cells (8). Adherence tohost cells is required for virulence of mucosal pathogens. Con-sequently, prevention of or interference with adherence of aparticular bacterial pathogen by molecules such as antibodiesto the adhesin prevent colonization and disease (33, 43). Forexample, multiple MAbs against the F41 adhesive fimbrialantigen of enterotoxigenic Escherichia coli (ETEC) protectedanimals against a challenge with F41-positive ETEC (56).

Multiple hemagglutinin genes have been cloned from P.gingivalis by functional screening (38, 50, 51). One of these, thegene coding for hemagglutinin A from P. gingivalis, has beenisolated and shown to contain four large direct repeats (25).When a P. gingivalis expression library was screened for cloneswhich bind human oral epithelial cells, all positive clones werefound to have DNA homology to hemagglutinin A (16). Thus,an immune response to HagA or other hemagglutinins mightprevent the colonization of P. gingivalis by inhibiting its adher-ence to oral tissues.

Vaccination against a disease may have both prophylacticand therapeutic value. Immunization with a vaccine containingkilled P. gingivalis suppresses the progress of experimental

periodontitis in Macaca fascicularis, suggesting that immuniza-tion against P. gingivalis may be an effective means of control-ling the disease (57). Unfortunately, vaccines based on killedbacteria can cause toxic reactions (42). Subunit vaccines mayreduce the problems associated with inactivated bacterial par-ticles because of their defined chemical and physical proper-ties. However, the production of adhesins for subunit vaccinesis often difficult due to contamination with other virulencefactors during the tedious process of purification. Other po-tential limitations include low levels of immunogenicity andfailure to induce the desired type of immune response com-pared with natural infection (45).

Oral administration of vaccines induces a secretory immu-noglobulin A (IgA) response upon absorption of the antigen bythe gut-associated lymphoid tissue (GALT) (41). The mostsuccessful vaccines developed against intracellular bacteriahave been based on replication-competent, avirulent or atten-uated bacteria such as the BCG strain of Mycobacterium bovis(19) and most of all Salmonella enterica serovar Typhimurium(9). Salmonella is an effective antigen delivery system to theGALT, which initiates production of specific secretory immu-noglobulin A for protection against pathogens that colonize orcross mucosal surfaces to initiate infection. This has been es-tablished as an effective means of stimulating significant levelsof specific mucosal secretory immunoglobulin A directedagainst a variety of heterologous antigens and has also beenshown to stimulate the production of serum antibodies andcell-mediated responses (7).

Salmonella vaccine strains expressing a streptococcal adhe-sin (24, 59), Listeria extracellular proteins (13, 28–30), a Leish-mania surface glycoprotein (40), the Campylobacter immuno-

* Corresponding author. Mailing address: Department of Oral Bi-ology, University of Florida, Box 100424 JHMHSC, Gainesville, FL32610-0424. Phone: (352) 392-5937. Fax: (352) 392-2361. E-mail:kozarov@dental.ufl.edu.

† Present address: Hoechst Marion Roussel Ltd., Minato Ward,Tokyo, Japan 107-8465.

732

on March 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

reactive transport protein (48), an Entamoeba protectiveantigen (60), the hepatitis B virus core antigen (54), the Bor-detella pertussis filamentous hemagglutinin (23), and P. gingi-valis hemagglutinin B (17) have been constructed. In this study,we sought to obtain and characterize an avirulent S. entericaserovar Typhimurium vaccine strain which expresses anotherpotentially useful immunogen of P. gingivalis that may conferfunctional protection from periodontal tissue destruction in-duced by P. gingivalis. Heterologous expression of hemagglu-tinin A was obtained in both E. coli and the S. enterica serovarTyphimurium avirulent vaccine strain x4072.

MATERIALS AND METHODS

Bacterial strains, plasmids, cell lines, and media. S. enterica serovar Typhi-murium x4072 SR-11 (Dcya Dcrp Dasd) was used as the vaccine strain. Theplasmid expression vector pYA292 is the vector component of this balanced-lethal system (21). A single copy of the asd gene per cell is sufficient for normalgrowth of S. enterica serovar Typhimurium Dasd, allowing the plasmid to bepresent in low copy numbers. E. coli x6097 (F2 D[lac-pro]rpsL DasdA4[zhf-2::Tn10]thi f80dlacZDM15), also with asd deleted, was used as a cloning host forpYA292-based constructs (44). E. coli JM109 [recA1 endA1 gyrA96 thi-1 hsdR17supE44 relA1 D(lac-proAB) (F9 traD36 proAB lacIqZDM15)] and E. coli DH5a(Life Technologies, Gaithersburg, Md.) were used for other routine cloningprocedures. Plasmid pHagA2 contains 8,585 bp of the hagA gene (25) cloned intothe XbaI-SacI sites of pBluescript II(1). Plasmid pYA292, E. coli x6097, and S.enterica serovar Typhimurium x4072 were kindly provided by Roy Curtiss III,Washington University, St. Louis, Mo.

Both S. enterica serovar Typhimurium and E. coli were cultured aerobically onLuria-Bertani medium (53) or on plates solidified with 1% agar, with the addi-tion of DL-a,ε-diaminopimelic acid (50 mg/ml) for the plasmid-free Dasd strains.pBluescript transformants were grown in medium supplemented with 100 mg ofampicillin per ml.

Recombinant DNA manipulations. Plasmids were isolated by the alkali lysismethod on purification columns (Qiagen, Santa Clarita, Calif.). RecombinantDNA techniques (restriction endonuclease digestion, DNA fragment purifica-tion, alkaline phosphatase treatment, and ligation) were essentially as describedpreviously (53) or as specified by the manufacturer. Restriction enzymes wereobtained from Promega Corp. (Madison, Wis.) or New England Biolabs (Bev-erly, Mass.), calf alkaline phosphatase was obtained from Boehringer Mannheim(Indianapolis, Ind.), and T4 DNA ligase was obtained from Life Technologies.Oligonucleotide synthesis was performed by Genosys (The Woodlands, Tex.).DNA sequencing was performed at the University of Florida InterdisciplinaryCenter for Biotechnology Research Core laboratory using ABI 373 and 377Perkin-Elmer/Applied Biosystems automated DNA sequencers. A RoboticWorkstation (ABI Catalyst 800) and a Perkin-Elmer Cetus PEC 9600 thermo-cycler were used in fluorescent cycle sequencing reactions. After adapter wasadded, the ligation mix was heated at 45°C for 5 min before the ligase and theligase buffer were added. The incubation proceeded overnight at 16°C. Chemi-cally competent E. coli x6097 was prepared by the method of Hanahan (26) inSOB medium supplemented with DAP. Electrocompetent S. enterica serovarTyphimurium x4072 cells were prepared as previously described (5) in thepresence of DAP.

PCR screening. Transformed E. coli x6097 colonies were screened by PCR(52) with four oligonucleotides to amplify an internal sequence of hagA and toconfirm the presence of the hagA insert in pYA292, as follows: ST2/1, 59-GCGGAATTCAGCTTCGATACGCAAACGCTTCCTAACG-39 corresponding tonucleotides 1070 to 1097 of the hagA coding strand (25); ST2/2, 59-CGATAACTGCAGTATTACGCAGGCAAATCTACCGTACGCTCGATCC-39 corre-sponding to nucleotides 4203 to 4231 of the hagA noncoding strand; PA2,59-GCGGATCCACCTTTTGAAAGTATTAAAGATTAATG-39 complemen-tary to bases 338 to 364; and TB4, 59-GGCTCGTATAATGTGTGGA-39 corre-sponding to nucleotides 257 to 239 upstream of the Met codon in pYA292 (21).In addition, to confirm the presence of the full-size hagA in the expressionplasmid pNM1.1, PCR was performed with primers flanking the gene, as follows:upstream oligonucleotide 208, 59-TTTCGCTCGCCGTCCTATTATC-39 corre-sponding to nucleotides 387 to 408 of the coding strand, and downstream oligo-nucleotide 207-2, 59-CGATCGGTTGGTAGAGCATAC-39 complementary tonucleotides 8273 to 8293 of the noncoding strand (25).

Before the reactions were performed the suitability of the primers was verifiedwith Oligo 4.0 software. The reactions were performed in a final volume of 50 mlcontaining 2.35 mM MgCl2, 0.3 mM each primer, 0.4 mM each deoxynucleosidetriphosphate (each), 1.25 U of Taq DNA polymerase (Promega), and 0.78 U ofPfu DNA polymerase (Stratagene, La Jolla, Calif.). Amplifications included aninitial denaturation step of 1 min at 94°C followed by 30 cycles each of 94°C for30 s, 55°C for 30 s, and 68°C for 6 min, with a final extension step of 10 min at72°C, using a PTC-100 thermal cycler (MJ Research, Watertown, Mass.).

Immunological techniques. Optimal dilutions of antibody, secondary antibodyconjugate, and color substrate were selected by a series of dot blots and Western

blots tested in multiwell incubation trays. Monoclonal antibody (MAb) 61BG1.3(IgG1 isotype), (kindly provided by Rudolf Gmur, Institute of Oral Microbiologyand General Immunology, Zurich, Switzerland [22]) was used to detect theexpression of the target protein. Serum from subcutaneously challenged mice(see Fig. 3B) was obtained as previously described (36). Immunodot blots wereused to detect HagA expression, as follows. Bacteria were collected, washed andresuspended in phosphate-buffered saline (PBS), and probe sonicated threetimes for 20 s on ice with a microsonicator (Kontes, Vineland, N.J.) in thepresence of Complete proteinase inhibitor (Boehringer Mannheim). After cen-trifugation at 16,000 3 g, the supernatant was collected for immunoanalysis. Forselection of transformants expressing the target protein, colonies were lifted ontoNitro ME nitrocellulose filters (MSI, Westboro, Mass.) or Protran BA83 (Schlei-cher & Schuell, Keene, N.H.) and colony immunoscreening was performed (53).

For Western immunoblots, 40-ml samples were mixed with 63 loading samplebuffer (100mM Tris [pH 6.8], 5% [wt/vol] sodium dodecyl sulfate [SDS], 50%glycerol, 7.5% b-mercaptoethanol, 0.00125% bromphenol blue) and were loadedonto 10 to 20% gradient polyacrylamide gels after incubation in a boiling-waterbath for 3 min. The gels were run in Tris-SDS buffer by the method of Laemmli(36a), and the proteins were reversibly visualized with a zinc staining kit (Bio-Rad, Hercules, Calif.). Broad-range molecular weight standards were used (Bio-Rad). After destaining, the proteins were transferred to a nitrocellulose mem-brane in a Trans Blot device (Bio-Rad) by standard procedures (3). Themembranes were blocked (the blocking solution consisted of 5% Carnation drynonfat milk and 0.02% sodium azide in Tris-buffered saline [TBS]) and reactedfor 1.5 h with MAb 61BG1.3 diluted 1:20 or with serum from mice orallyimmunized by gastric intubation with recombinant Salmonella vaccine diluted1:500 in the blocking solution (1% nonfat milk and 0.02% azide in TBS). Thesecondary antibody, alkaline phosphatase-conjugated goat anti-mouse IgG(Fisher), at a 1:500 dilution in the blocking solution, was applied for 1 h.Developing tablets (Sigma, St. Louis, Mo.) containing (after being dissolved)0.01% nitroblue tetrazolium and 0.016% 5-bromo-4-chloro-3-indolyl phosphate(the color substrate) were used to develop the blots. Protein concentrations weredetermined with the bicinchoninic assay reagents (Sigma) as specified by themanufacturer. Proteins on blotted membranes were reversibly visualized withPonceau S solution (Sigma).

Mouse immunization. For oral immunization, a single colony of Salmonellavaccine strain was grown in Luria-Bertani broth at 37°C. Western analysis of thestrain was done prior to immunization to confirm the presence of HagA in thecell lysate. After centrifugation at 5,000 3 g, the bacterial pellet was resuspendedin 0.1 M NaHCO3 to yield ;1010CFU/ml. Female BALB/c mice, 8 to 10 weeksold, were obtained from Charles River Laboratories, Inc. (Bar Harbor, Maine).The mice were intubated twice with 0.1 ml of Salmonella suspension at a 2-weekinterval. Serum was collected 9 weeks after the boost.

Human subject sera. For immunoanalysis, serum from a clinical periodontalpatient was obtained as described previously (39). The protocol for using adulthuman subjects was reviewed and approved by the University of Florida Insti-tutional Review Committee. The patient was diagnosed with adult group 2periodontitis (39). In this group, the alveolar bone loss is $8 mm at no more thanone site and there are any number of sites with 4 to 5.9 mm of bone loss. Theserum used contains anti-P. gingivalis antibodies at the following titers: anti-P.gingivalis 33277, 279 mg/ml; anti-P. gingivalis 381, 371 mg/ml; and anti-P. gingivalisW83, 103 mg/ml. For age-matched normal controls, the mean numbers are 20, 17,and 5 mg/ml, respectively.

IEM. To detect a previously identified HagA-specific epitope (22) in theSalmonella vaccine strain, the vaccine cells were examined by immunoelectronmicroscopy (IEM) at the Electron Microscopy Core Laboratory of the Interdis-ciplinary Center for Biotechnology Research, University of Florida. Whole cellsfrom the analyzed strains were collected from freshly grown liquid cultures.Minimum fixation was used to preserve the native conformation of the antigenicdeterminants. MAb 61BG1.3 was used as the primary antibody, and unrelatedmouse MAb of matching isotype was used as a control. All samples were prela-beled and postlabeled after embedding and cutting of thin sections.

For prelabeling of bacteria with MAb 61BG1.3, the bacterial samples werepelleted by centrifugation, washed in PBS (pH 7.2), and fixed for 15 min in 4%paraformaldehyde. After fixation, the samples were washed twice in PBS. Eachsample was split into two equal parts, and each part was incubated for 10 minwith 1% bovine serum albumin. The samples were centrifuged, the supernatantwas removed, and 0.25-ml volumes of hybridoma cultures containing either themouse MAb 61BG1.3 or an unrelated IgG1 isotype-matched control mouseMAb were diluted 1:20 with PBS and added to the pellets. The pellets wereresuspended and incubated overnight at 4°C.

For embedding of prelabelled bacteria, samples were pelleted and washedthree times in PBS, dehydrated in an ethanol series to 100% ethanol, and theninfiltrated and embedded in Unicryl.

For postlabeling of Unicryl sections of the bacteria samples with MAb61BG1.3, thin sections of Unicryl-embedded samples on Formvar-coated nickelgrids were incubated on 10-ml drops of 1% milk in high-salt Tween buffer (HST)(pH 7.2) for 10 min. The grids were blotted with filter paper and placed on eitherMAb 61BG1.3 or the control MAb which had been diluted 1:20 in HST. Afterthe grids were incubated with the antibody overnight at 4°C in a moist environ-ment, they were washed twice for 10 min each in HST buffer and once in PBS.They were then incubated for 1 h at room temperature on drops of anti-mouse

VOL. 68, 2000 SALMONELLA VACCINE TO P. GINGIVALIS 733

on March 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

IgG (Jackson ImmunoResearch Laboratories, Inc.) labeled with 18-nm-diametergold particles, diluted 1:40 in PBS, and centrifuged before use. Finally the gridswere washed three times for 10 min each in PBS, incubated on Trump fixative for10 min, washed in distilled water, poststained with uranyl acetate and leadcitrate, and examined under a Hitachi 7000 transmission electron microscope.

RESULTS

Construction of the HagA expression plasmid pNM1.1. Toconstruct a plasmid for heterologous expression of HagA,hagA (25), including its upstream and downstream regulatoryregions, was obtained from pHagA2 by digestion with SacI andSalI and ligated into the SalI site of pYA292 by using a syn-thetic SalI-SacI adapter (Fig. 1). E. coli x6097 cells trans-formed with this construct were grown on Luria-Bertani agarplates in the presence of 5-bromo-4-chloro-3-indolyl-b-D-ga-lactopyranoside (X-Gal) (40 mg/ml). White colonies werescreened by PCR for the presence of a hagA insert as describedin Materials and Methods. Plasmid pNM1, with the expectedsize, was isolated from a positive colony, and the presence ofthe hagA insert was confirmed by PCR with mixed pairs ofprimers (vector plus insert): ST2/1-ST2/2 and ST2/2-TB4primer pairs were used for positive reactions, and the ST2/1-TB4 pair was used for the negative control (a graphic repre-sentation of the oligonucleotides is given in Fig. 1). The pres-ence and orientation of the insert were also confirmed bysequencing reactions with TB4 and PA2 primers. Analysis ofthe expression plasmid pNM1 with primers flanking the full-size 8-kb insert demonstrated the presence of hagA (data notshown).

Construction of the recombinant S. enterica serovar Typhi-murium x4072 vaccine strain. pNM1 was reisolated and trans-ferred to S. enterica serovar Typhimurium x4072 by electropo-ration. To screen S. enterica serovar Typhimurium x4072 fortransformants which expressed HagA, colony immunoscreen-ing on 30 transformants was performed. Both E. coli x6097 and

S. enterica serovar Typhimurium x4072 were transformed withthe pNM1 expression construct. Screening of 30 S. entericaserovar Typhimurium colonies by enzyme-linked immunode-tection of HagA with the 61BG1.3 monoclonal antibody re-sulted in the identification of six HagA-positive S. entericaserovar Typhimurium transformants. A plate with E. coli x6097transformants was used as a positive control. This immunode-tection assay revealed that only 20% of Salmonella transfor-mants expressed HagA compared to 100% of the control E.coli x6097 transformants. Five of the S. enterica serovar Typhi-murium HagA-positive transformants were grown, and plas-mid DNA preparations (pNM1.1 to pNM1.5) were made andanalyzed by PCR with internal and mixed pairs of primers, asdescribed for the initial screening for pNM1. The size of eachof these five plasmids was found to be equal to that of the E.coli-derived original. One of them (pNM1.1) was chosen forfurther study. Plasmid DNA was isolated from pNM1.1, andthe insert was sequenced with TB4 and PA2 primers. Theresults confirmed the presence in S. enterica serovar Typhi-murium of sequences identical to those of the plasmid (pNM1)isolated from E. coli.

Immunochemical analyses. SDS-polyacrylamide gel electro-phoresis and Western blot analyses were performed with MAb61BG1.3 to detect the expression of HagA in the S. entericaserovar Typhimurium x4072 vaccine strain (Fig. 2A, lane 5)compared to its expression in this strain containing vector only(lane 6) and in E. coli (lanes 1 and 3) compared to E. colicontaining the vectors only (lanes 2 and 4). The HagA epitope,recognized by MAb 61BG1.3 (22), was detected in the Salmo-nella vaccine strain and in both recombinant E. coli strainsused, JM109 (lane 1) and x6097, the cloning host for Asd1

plasmid pYA292-based constructs (lane 3). The MAb was notreactive with the control Salmonella strain containing the vec-tor only (lane 6). Interestingly, the level of expression from the

FIG. 1. Construction of pNM1 (see Materials and Methods and Results). The hagA coding sequence including flanking regions was cloned in pYA292 by using aSalI-SacI adapter. Striped line, hagA open reading frame; dotted boxes, hagA flanking sequences; arrows, primers used for PCR analysis. Not to scale.

734 KOZAROV ET AL. INFECT. IMMUN.

on March 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

same expression plasmid, pNM1.1, appeared to be lower in E.coli than in S. enterica serovar Typhimurium (lanes 3 and 5).

To test the immunogenicity of HagA expressed by the vac-cine strain, immune mouse serum was assayed for antibody toHagA by Western immunoblotting of a previously cloned 100-kDa recombinant HagA fragment (E. Kozarov and A. Progul-ske-Fox, unpublished data). The presence of specific anti-HagA antibodies in the serum was clearly demonstrated in Fig.2B, lane 2. A 1-mg sample of protein was loaded in each oflanes 1 and 2. As a control, a similar blot was reacted with thespecific anti-HagA MAb (lane 1).

To determine if the vaccine-derived HagA can be recog-nized by human antibodies, serum from a periodontitis patientwas also analyzed by Western blotting. As shown in Fig. 2C, ahigh-molecular-weight band is visible in the vaccine strainpreparation (lane 1) as compared to the Salmonella vectorcontrol (lane 3). Reactive antigen bands are also evident withthe positive control, lysed P. gingivalis cells (lane 2). The pres-ence of a unique protein species from the hemagglutinin-ex-pressing vaccine strain that reacts with the serum from theperiodontitis patient (lane 1) and is absent from the control(lane 3) is clearly evident.

Immunoelectron microscopy. To determine the cellular lo-cation of the expressed HagA antigen, immunoelectron mi-croscopy was performed on the S. enterica serovar Typhi-murium x4072(pNM1.1) vaccine strain (Fig. 3A), S. entericaserovar Typhimurium x4072 (Fig. 3C, vector-only control), andP. gingivalis 381 (Fig. 3D, positive control). These results dem-onstrate the expression of the antigen in the vaccine strain.Figures 3D and E are positive and negative controls respec-tively.

DISCUSSION

Periodontitis in humans is thought to be caused by a groupof predominantly gram-negative anaerobic bacteria, amongwhich P. gingivalis is prominent. Considerable scrutiny is re-quired to select useful immunogens that can elicit functionalprotection against periodontal tissue destruction induced byoral microorganisms that already colonize or infect the host(31). Immunization with a vaccine containing killed whole cellsof P. gingivalis suppresses the progress of experimental peri-odontitis in M. fascicularis (57). However, a vaccine composedonly of specific protective antigens is most desirable.

Hemagglutinin A is the largest member of a family of P.gingivalis proteins, including hemagglutinins A and D, whosegenes were isolated via functional screening for hemaggluti-nating activity (25, 50). They have extensive homology to each

other and to other abundant P. gingivalis proteins includingprotease PrtP (4), PrtH (20), protease RGP-1 (47), proteasePrtR (35), argingipain (46), and Arg1 (11). One of the four;450-amino-acid (aa) repeats making up more than half of theHagA polypeptide is the common shared motif. The MAb usedhere for detection of expression of HagA, 61BG1.3 (22), pro-vides passive protection against recolonization of P. gingivalisin humans (6) and recognizes an epitope present in HagA andin the proteins to which HagA has homology. With five copiesof the epitope, HagA itself is a multivalent carrier. The61BG1.3 epitope may be a component of a binding domaincommon to multiple gene products of this organism and maythus represent a functionally important target of the specificimmune response of the host to P. gingivalis (11). The existenceof multiple gene products containing a common epitope haspreviously been reported for Moraxella catarrhalis. The high-molecular-weight UspA protein of M. catarrhalis is present onthe surface of all M. catarrhalis disease isolates examined todate and contains the epitope for a MAb (MAb 17C7) whichenhances the pulmonary clearance of this organism in a mousemodel system (27). Recently, the presence of a second M.catarrhalis gene, uspA2, which also encodes the MAb 17C7-reactive epitope, has also been reported (1). Interestingly, bothUspA1 and UspA2 proteins closely resemble adhesins pro-duced by other bacterial pathogens. With P. gingivalis, thehemagglutinating adhesin HagA (25) shares a 25-aa residueprotective epitope found in the arginine-specific protease (11)and the lysine-specific protease (51) of this organism. Thus,construction of a vehicle for delivering the HagA-encodedantigens may be an efficient way of eliciting an immune re-sponse capable of preventing colonization of P. gingivalis inhumans.

The apparent processing of the HagA polypeptide (Fig. 2A,lane 5) may be because many P. gingivalis gene products areposttranslationally processed to contribute to the formation ofmultimeric surface protein-adhesin complexes (37). It is estab-lished that various cell surface and secretory proteins are pro-cessed in P. gingivalis (34).

The epitopes recognized by sera from periodontitis patientshave been previously reported to fall within the beta subunit, ahemagglutinin and/or adhesin component of the arginine-spe-cific proteases of P. gingivalis (ArgI, ArgIA, and ArgIB) (11).The antibody response in animals to a protease carrying bothcatalytic and hemagglutinating domains is confined only to theadhesive part of the protein, suggesting that the catalytic por-tion is not exposed (J. Travis, personal communication). Thus,a HagA vaccine which includes an epitope common to a family

FIG. 2. (A) Western immunoblot analysis of soluble cellular fractions of S. enterica serovar Typhimurium and E. coli with HagA-specific MAb 61BG1.3. Lanes: 1,E. coli(pHagA1); 2, E. coli(pUC18); 3, E. coli(pNM1); 4, E. coli(pYA292); 5, S. enterica serovar Typhimurium(pNM1.1); 6, S. enterica serovar Typhimurium(pYA292);7, P. gingivalis 381. (B) Western immunoblot of recombinant HagA with HagA-specific MAb 61BG1.3 as the primary antibody (lane 1) and sera from Salmonella-immunized mice (lane 2). (C) Western blotting with serum from a periodontitis patient. Lanes 1: S. enterica serovar Typhimurium(pNM1.1); 2, P. gingivalis 381; 3, S.enterica serovar Typhimurium(pYA292). Molecular masses are given in kilodaltons.

VOL. 68, 2000 SALMONELLA VACCINE TO P. GINGIVALIS 735

on March 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

FIG. 3. Transmission electron micrographs of strains used in this study. (A) S. enterica serovar Typhimurium x4072(pNM1.1) vaccine strain incubated with MAb61BG1.3. (B) Same strain reacted with control unrelated antibody. (C) S. enterica serovar Typhimurium x4072(pYA292) vector-only control reacted with MAb61BG1.3. (D) P. gingivalis 381 reacted with MAb 61BG1.3 (positive control). (E) Same as panel D, with an unrelated antibody.

736

on March 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

of hemagglutinins in addition to proteases may be an effectiveimmunogen against a variety of virulence factors.

The goal of the present effort was to determine if HagA canbe expressed in an immunogenic form in a Salmonella-basedlive vaccine strain. In this study we show that hemagglutinin A,a 2,628-aa P. gingivalis protein which agglutinates erythrocytesand is implicated in the virulence of the bacterium, can beexpressed in an attenuated Salmonella vaccine strain. Serumfrom mice immunized with the vaccine strain react with puri-fied HagA. In addition, we have demonstrated that the HagAantigen of the vaccine strain is recognized by antibodies in theserum of a periodontal patient.

The live vaccine strain, S. enterica serovar Typhimuriumx4072, is both avirulent and immunogenic but retains its abilityto colonize the GALT (12). This vaccine strain has been usedpreviously to express another hemagglutinin from P. gingivalis(18). The nonfused filamentous hemagglutinin of Bordetellapertussis, an important adhesin in the early interactions be-tween the bacterium and host cells, has also been efficientlyexpressed in S. enterica serovar Typhimurium (23). PlasmidpYA292 has also been used to express streptococcal surfaceantigens (15), Entamoeba hystolytica antigens (10), and hepa-titis B virus antigens (54).

The presence of the hagA gene-associated protein in Salmo-nella is demonstrated by immunoanalysis. This suggests thatalthough no E. coli-like ribosome binding sequence is presentin the 59-untranslated region of hagA, the E. coli and Salmo-nella transcription and translation machinery still functions toexpress this gene. Multiple protein bands are recognized inboth E. coli and P. gingivalis in these blots, which suggests thatthe 2,628-aa target protein is being processed by proteases.

In gram-negative bacteria, many periplasmic and outermembrane preproteins have a signal sequence at the N termi-nus which is cleaved during translocation of the proteinthrough the cytoplasmic membrane. By using the PSORT al-gorithm (58), the structure of the N-terminal region of HagAis predicted to be typical of a prokaryotic signal peptide, initi-ating inner membrane transfer of the precursor. It consists ofpositive N-terminal charges, a central hydrophobic region, andan Ala signal peptidase cleavage site (Fig. 4). Cleavage at thepredicted cleavage sites would result in an outer membrane-embedded or secreted protein.

In addition to being surface exposed, HagA is probablyreleased from the cells since hemagglutination activity in theculture medium has been reported for P. gingivalis 381 (32). Inour study, Western immunoblotting of spent culture mediumfrom different P. gingivalis strains demonstrated an abundanceof protein species recognized by the protective antibody (datanot shown). These proteins may either be independently re-leased or constitute a component of blebs, i.e., 100-nm mem-brane vesicles released by P. gingivalis. Proteins secreted byMycobacterium, another mucosal pathogen, have also beensuggested to be major immune targets (2). Secreted proteinsare preferentially recognized by T cells before somatic proteins(14), and it has been shown that secreted or surface-localizedantigens in Salmonella display superior efficacy over that ofsomatic display (29, 55). Accordingly, by using IEM we dem-

onstrated surface expression of the target protein in Salmo-nella. An immune response to HagA may be efficient againstdifferent strains of the pathogen. In addition, MAb 1A1, whichrecognizes the same epitope as MAb 61BG1.3, strongly inhib-its the agglutination of human erythrocytes by P. gingivalisculture supernatant (11). These findings suggests that Salmo-nella expressing HagA would be a good choice as a live-vaccinecandidate.

In conclusion, heterologous expression of hemagglutinin A,a major virulence-associated surface protein of P. gingivalis,was demonstrated in an avirulent vaccine strain of S. entericaserovar Typhimurium. A balanced-lethal non-antibiotic-resis-tant expression vector for the Salmonella host system was usedfor the expression. Successful delivery of the target protein viathe mucosal immune system was demonstrated by the presenceof antibodies in the sera of mice which had received the vac-cine strain by gastric intubation. A well-characterized epitopeof the HagA protein (22) was shown to be present in its nativeconfiguration by immunochemistry and IEM analysis of thevaccine strain. The presence of hemagglutinin A on the Sal-monella surface was demonstrated by IEM. In addition, theHagA antigen of the vaccine strain was recognized by antibod-ies present in the serum of a human periodontitis patient.Finally, by using purified recombinant HagA, the presence ofspecific anti-hemagglutinin A antibodies in the serum of orallyimmunized mice was established. Therefore, testing of thisvaccine construct for the elicitation of a protective immuneresponse is continuing.

ACKNOWLEDGMENTS

We thank L. Jeannine Brady for critical review of the manuscript.This work was supported by NIH grant DE07496 to A. Progulske-

Fox.

REFERENCES

1. Aebi, C., I. MacIver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas,G. H. McCracken, Jr., and E. J. Hansen. 1997. A protective epitope ofMoraxella catarrhalis is encoded by two different genes. Infect. Immun. 65:4367–4377.

2. Andersen, P. 1994. Effective vaccination of mice against Mycobacteriumtuberculosis infection with a soluble mixture of secreted mycobacterial pro-teins. Infect. Immun. 62:2536–2544.

3. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.Smith, and K. Struhl. 1997. Current protocols in molecular biology, vol. 1.Greene Publishing Associates and Wiley-Interscience, New York, N.Y.

4. Barkocy-Gallagher, G. A., N. Han, J. M. Patti, J. Whitlock, A. Progulske-Fox, and M. S. Lantz. 1996. Analysis of the prtP gene encoding porphypain,a cysteine proteinase of Porphyromonas gingivalis. J. Bacteriol. 178:2734–2741.

5. Binotto, J., P. R. MacLachlan, and K. E. Sanderson. 1991. Electrotransfor-mation in Salmonella typhimurium LT2. Can. J. Microbiol. 37:474–477.

6. Booth, V., F. P. Ashley, and T. Lehner. 1996. Passive immunization withmonoclonal antibodies against Porphyromonas gingivalis in patients with pe-riodontitis. Infect. Immun. 64:422–427.

7. Cardenas, L., and J. D. Clements. 1992. Oral immunization using live atten-uated Salmonella spp. as carriers of foreign antigens. Clin. Microbiol. Rev.5:328–342.

8. Chandad, F., and C. Mouton. 1990. Molecular size variation of the hemag-glutinating adhesin HA-Ag2, a common antigen of Bacteroides gingivalis.Can. J. Microbiol. 36:690–696.

9. Chatfield, S. N., and G. Dougan. 1997. Attenuated Salmonella as a live vectorfor expression of foreign antigens, p. 331–361. In M. M. Levine, G. C.

FIG. 4. N-terminal amino acids of HagA. 1, basic-type residue; H, hydrophobic-type residue; P, polar uncharged-type residue.

VOL. 68, 2000 SALMONELLA VACCINE TO P. GINGIVALIS 737

on March 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Woodrow, J. B. Kaper, and G. S. Cobon (ed.), New generation vaccines.Marcel Dekker, Inc., New York, N.Y.

10. Cieslak, P. R., T. Zhang, and S. L. Stanley, Jr. 1993. Expression of arecombinant Entamoeba histolytica antigen in a Salmonella typhimurium vac-cine strain. Vaccine 11:773–776.

11. Curtis, M. A., J. Aduse-Opoku, J. M. Slaney, M. Rangarajan, V. Booth, J.Cridland, and P. Shepherd. 1996. Characterization of an adherence andantigenic determinant of the ArgI protease of Porphyromonas gingivaliswhich is present on multiple gene products. Infect. Immun. 64:2532–2539.

12. Curtiss, R., III, and S. M. Kelly. 1987. Salmonella typhimurium deletionmutants lacking adenylate cyclase and cyclic AMP receptor protein areavirulent and immunogenic. Infect. Immun. 55:3035–3043.

13. Darji, A., C. A. Guzman, B. Gerstel, P. Wachholz, K. N. Timmis, J. Wehland,T. Chakraborty, and S. Weiss. 1997. Oral somatic transgene vaccinationusing attenuated S. typhimurium. Cell 91:765–775.

14. Daugelat, S., C. H. Ladel, B. Schoel, and S. H. Kaufmann. 1994. Antigen-specific T-cell responses during primary and secondary Listeria monocyto-genes infection. Infect. Immun. 62:1881–1888.

15. Doggett, T. A., E. K. Jagusztyn-Krynicka, and R. Curtiss III. 1993. Immuneresponses to Streptococcus sobrinus surface protein antigen A expressed byrecombinant Salmonella typhimurium. Infect. Immun. 61:1859–1866.

16. Duncan, M. J., S. A. Emory, and E. C. Almira. 1996. Porphyromonas gingi-valis genes isolated by screening for epithelial cell attachment. Infect. Im-mun. 64:3624–3631.

17. Dusek, D. M., A. Progulske-Fox, and T. A. Brown. 1994. Systemic andmucosal immune responses in mice orally immunized with avirulent Salmo-nella typhimurium expressing a cloned Porphyromonas gingivalis hemaggluti-nin. Infect. Immun. 62:1652–1657.

18. Dusek, D. M., A. Progulske-Fox, J. Whitlock, and T. A. Brown. 1993. Isola-tion and characterization of a cloned Porphyromonas gingivalis hemagglutininfrom an avirulent strain of Salmonella typhimurium. Infect. Immun. 61:940–946.

19. Fennely, G. J., W. R. Jacobs, Jr., and B. R. Bloom. 1997. BCG as a recom-binant vaccine vector, p. 363–377. In M. M. Levine, G. C. Woodrow, J. B.Kaper, G. S. Cobon (ed.), New Generation Vaccines. Marcel Dekker, Inc.,New York, N.Y.

20. Fletcher, H. M., H. A. Schenkein, and F. L. Macrina. 1994. Cloning andcharacterization of a new protease gene (prtH) from Porphyromonas gingi-valis. Infect. Immun. 62:4279–4286.

21. Galan, J. E., K. Nakayama, and R. Curtiss III. 1990. Cloning and charac-terization of the asd gene of Salmonella typhimurium: use in stable mainte-nance of recombinant plasmids in Salmonella vaccine strains. Gene 94:29–35.

22. Gmur, R., G. Werner-Felmayer, and B. Guggenheim. 1988. Production andcharacterization of monoclonal antibodies specific for Bacteroides gingivalis.Oral Microbiol. Immunol. 3:181–186.

23. Guzman, C. A., M. J. Walker, M. Rohde, and K. N. Timmis. 1991. Directexpression of Bordetella pertussis filamentous hemagglutinin in Escherichiacoli and Salmonella typhimurium aroA. Infect. Immun. 59:3787–3795.

24. Hajishengallis, G., E. Harokopakis, S. K. Hollingshead, M. W. Russell, andS. M. Michalek. 1996. Construction and oral immunogenicity of a Salmonellatyphimurium strain expressing a streptococcal adhesin linked to the A2/Bsubunits of cholera toxin. Vaccine 14:1545–1548.

25. Han, N., Whitlock J., Progulske-Fox A. 1996. The hemagglutinin gene A(hagA) of Porphyromonas gingivalis 381 contains four large, contiguous, di-rect repeats. Infect. Immun. 64:4000–4007.

26. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plas-mids. J. Mol. Biol. 166:557–580.

27. Helminen, M. E., I. MacIver, J. L. Latimer, J. Klesney-Tait, L. D. Cope, M.Paris, G. H. McCracken, Jr., and E. J. Hansen. 1994. A large, antigenicallyconserved protein on the surface of Moraxella catarrhalis is a target forprotective antibodies. J. Infect. Dis. 170:867–872.

28. Hess, J., G. Dietrich, I. Gentschev, D. Miko, W. Goebel, and S. H. Kauf-mann. 1997. Protection against murine listeriosis by an attenuated recom-binant Salmonella typhimurium vaccine strain that secretes the naturallysomatic antigen superoxide dismutase. Infect. Immun. 65:1286–1292.

29. Hess, J., I. Gentschev, D. Miko, M. Welzel, C. Ladel, W. Goebel, and S. H.Kaufmann. 1996. Superior efficacy of secreted over somatic antigen displayin recombinant Salmonella vaccine induced protection against listeriosis.Proc. Natl. Acad. Sci. USA 93:1458–1463.

30. Hess, J., I. Gentschev, G. Szalay, C. Ladel, A. Bubert, W. Goebel, and S. H.Kaufmann. 1995. Listeria monocytogenes p60 supports host cell invasion byand in vivo survival of attenuated Salmonella typhimurium. Infect. Immun.63:2047–2053.

31. Holt, S. C., M. Brunsvold, A. Jones, R. Wood, and J. L. Ebersole. 1995. Cellenvelope and cell wall immunization of Macaca fascicularis: effect on theprogression of ligature-induced periodontitis. Oral Microbiol. Immunol. 10:321–333.

32. Inoshita, E., A. Amano, T. Hanioka, H. Tamagawa, S. Shizukuishi, and A.Tsunemitsu. 1986. Isolation and some properties of exohemagglutinin fromthe culture medium of Bacteroides gingivalis. Infect. Immun. 57:421–427.

33. Johnson, J. R. 1991. Virulence factors in Escherichia coli urinary tract infec-

tion. Clin. Microbiol. Rev. 4:80–128.34. Kadowaki, T., K. Nakayama, F. Yoshimura, K. Okamoto, N. Abe, and K.

Yamamoto. 1998. Arg-gingipain acts as a major processing enzyme for var-ious cell surface proteins in Porphyromonas gingivalis. J. Biol. Chem. 273:29072–29076.

35. Kirszbaum, L., C. Sotiropolos, S. C. Jackson, N. Slakeski, and E. C. Reyn-olds. 1995. Complete nucleotide sequence of a gene prtR of Porphyromonasgingivalis W50 encoding a 132 kDa protein that contains an arginine-specificthiol endopeptidase domain and a hemagglutinin domain. Biochem. Byo-phys. Res. Commun. 207:424–431.

36. Kozarov, E., J. Whitlock, H. Dong, E. Carrasco, and A. Progulske-Fox. 1998.The number of direct repeats in hagA is variable among Porphyromonasgingivalis strains. Infect. Immun. 66:4721–4725.

36a.Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

37. Lamont, R. J., and H. F. Jenkinson. 1998. Life below the gum line: patho-genic mechanisms of Porphyromonas gingivalis. Microbiol. Mol. Biol. Rev.62:1244–1263.

38. Lepine, G., and A. Progulske-Fox. 1996. Duplication and differential expres-sion of hemagglutinin genes in Porphyromonas gingivalis. Oral Microbiol.Immunol. 11:65–78.

39. McArthur, W. P., C. Bloom, M. Taylor, J. Smith, T. Wheeler, and N. I.Magnusson. 1995. Antibody responses to suspected periodontal pathogensin elderly subjects with periodontal disease. J. Clin. Periodontol. 22:842–849.

40. McSorley, S. J., D. Xu, and F. Y. Liew. 1997. Vaccine efficacy of Salmonellastrains expressing glycoprotein 63 with different promoters. Infect. Immun.65:171–178.

41. Mestecky, J. 1987. The common mucosal immune system and the currentstrategies for induction of immune responses in external secretions. J. Clin.Immunol. 7:265–276.

42. Mortimer, E. A. 1988. Pertussis vaccine, p. 74–97. In S. A. Plotkin and E. A.Mortimer (ed.), Vaccines. The W. B. Saunders Co., Philadelphia, Pa.

43. Mouricout, M. 1991. Swine and cattle enterotoxigenic Escherichia coli-me-diated diarrhea. Development of therapies based on inhibition of bacteria-host interactions. Eur. J. Epidemiol. 7:588–604.

44. Nakayama, K., S. M. Kelly, and R. Curtiss III. 1988. Construction of anAsd1 expression-cloning vector: stable maintenance and high level expres-sion of cloned genes in a Salmonella vaccine strain. Bio/Technology 6:693–697.

45. Newman, M. J., and M. F. Powell. 1995. Immunological and formulationdesign considerations for subunit vaccines, p. 1–42. In M. F. Powell and M. J.Newman (ed.), Vaccine design: the subunit and adjuvant approach. PlenumPress, New York, N.Y.

46. Okamoto, K., Y. Misumi, T. Kadowaki, M. Yoneda, K. Yamamoto, and Y.Ikehara. 1995. Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor fromPorphyromonas gingivalis. Arch. Biochem. Biophys. 316:917–925.

47. Pavloff, N., J. Potempa, R. N. Pike, V. Prochazka, M. C. Kiefer, J. Travis, andP. J. Barr. 1995. Molecular cloning and structural characterization of theArg-gingipain proteinase of Porphyromonas gingivalis. Biosynthesis as aproteinase-adhesin polyprotein. J. Biol. Chem. 270:1007–1010.

48. Pawelec, D., E. Rozynek, J. Popowski, and E. K. Jagusztyn-Krynicka. 1997.Cloning and characterization of a Campylobacter jejuni 72Dz/92 gene encod-ing a 30 kDa immunopositive protein, component of the ABC transportsystem; expression of the gene in avirulent Salmonella typhimurium. FEMSImmunol. Med. Microbiol. 19:137–150.

49. Pike, R., W. McGraw, J. Potempa, and J. Travis. 1994. Lysine- and arginine-specific proteinases from Porphyromonas gingivalis. Isolation, characteriza-tion, and evidence for the existence of complexes with hemagglutinins.J. Biol. Chem. 269:406–411.

50. Progulske-Fox, A., S. Tumwarson, and S. C. Holt. 1989. The expression andfunction of Bacteroides gingivalis hemagglutinin gene in Escherichia coli. OralMicrobiol. Immunol. 4:121–131.

51. Progulske-Fox, A., S. Tumwasorn, G. Lepine, J. Whitlock, D. Savett, J. J.Ferretti, and J. A. Banas. 1995. The cloning, expression and sequence anal-ysis of a second Porphyromonas gingivalis gene that codes for a proteininvolved in hemagglutination. Oral Microbiol. Immunol. 10:311–318.

52. Runnebaum, I. B., P. Syka, and S. Sukumar. 1991. Vector PCR. BioTech-niques 11:446–448, 450–452.

53. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed., vol. 2. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

54. Schodel, F., S. Kelly, S. Tinge, S. Hopkins, D. Peterson, D. Milich, and R.Curtiss III. 1996. Hybrid hepatitis B virus core antigen as a vaccine carriermoiety. II. Expression in avirulent Salmonella spp. for mucosal immuniza-tion. Adv. Exp. Med. Biol. 397:15–21.

55. Stahl, S., and M. Uhlen. 1997. Bacterial surface display: trends and progress.Trends Biotechnol. 15:185–192.

56. van Zijderveld, F. G., A. M. van Zijderveld-van Bemmel, and D. Bakker.1998. The F41 adhesin of enterotoxigenic Escherichia coli: inhibition ofadhesion by monoclonal antibodies. Vet. Q. 20(Suppl)3:S73–S78.

57. Vasel, D., T. J. Sims, B. Bainbridge, L. Houston, R. Darveau, and R. C. Page.

738 KOZAROV ET AL. INFECT. IMMUN.

on March 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

1996. Shared antigens of Porphyromonas gingivalis and Bacteroides forsythus.Oral Microbiol. Immunol. 11:226–235.

58. von Heijne, G. 1986. A new method for predicting signal sequence cleavagesites. Nucleic Acids Res. 14:4683–4690.

59. Yamamoto, M., L. S. McDaniel, K. Kawabata, D. E. Briles, R. J. Jackson,J. R. McGhee, and H. Kiyono. 1997. Oral immunization with PspA elicits

protective humoral immunity against Streptococcus pneumoniae infection.Infect. Immun. 65:640–644.

60. Zhang, T., and S. L. Stanley, Jr. 1997. Expression of the serine rich Enta-moeba histolytica protein (SREHP) in the avirulent vaccine strain Salmonellatyphi TY2 chi 4297 (Dcya Dcrp Dasd): safety and immunogenicity in mice.Vaccine 15:1319–1322.

Editor: D. L. Burns

VOL. 68, 2000 SALMONELLA VACCINE TO P. GINGIVALIS 739

on March 1, 2020 by guest

http://iai.asm.org/

Dow

nloaded from