Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0216—Bacteriodetes, e.g. Bacteroides, Ornithobacter, Porphyromonas
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55544—Bacterial toxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• This invention relates to an oral composition and an immunogenic composition for the suppression of the pathogenic effects of the intra-oral bacterium Porphyromonas gingivalis associated with periodontal disease. It also relates to diagnostic tests for the presence of Porphyromonas gingivalis in subgingival plaque samples and specific antibodies against P. gingivalis antigens in sera.
• the compositions comprise synthetic peptide constructs corresponding to structurally and functionally significant areas of the PrtR-PrtK proteinase-adhesin complex of Porphyromonas gingivalis. Also disclosed are methods for preparing the synthetic peptide constructs.
• the synthetic peptide constructs are useful as immunogens in raising an immune response against P. gingivalis and can be used to generate protein-specific and peptide-specific antisera useful for passive immunization and as reagents for diagnostic assays.
• Periodontal diseases are bacterial-associated inflammatory diseases of the supporting tissues of the teeth and range from the relatively mild form of gingivitis, the non-specific, reversible inflammation of gingival tissue to the more aggressive forms of periodontitis which are characterised by the destruction of the tooth's supporting structures.
• Periodontitis is associated with a subgingival infection of a consortium of specific Gram-negative bacteria that leads to the destruction of the periodontium and is a major public health problem.
• One bacterium that has attracted considerable interest is Porphyromonas gingivalis as the recovery of this microorganism from adult periodontitis lesions can be up to 50% of the subgingival anaerobically cultivable flora, whereas P.
• gingivalis is rarely recovered, and then in low numbers, from healthy sites.
• a proportional increase in the level of P. gingivalis in subgingival plaque has been associated with an increased severity of periodontitis and eradication of the microorganism from the cultivable subgingival microbial population is accompanied by resolution of the disease.
• the progression of periodontitis lesions in non-human primates has been demonstrated with the subgingival implantation of P. gingivalis.
• P. gingivalis is a black-pigmented, anaerobic, proteolytic Gram-negative rod that obtains energy from the metabolism of specific amino acids.
• the microorganism has an absolute growth requirement for iron, preferentially in the form of heme or its Fe(III) oxidation product hemin and when grown under conditions of excess hemin is highly virulent in experimental animals.
• a number of virulence factors have been implicated in the pathogenicity of P. gingivalis including the capsule, adhesins, cytotoxins and extracellular hydrolytic enzymes.
• In order to develop an efficacious and safe vaccine to prevent P. gingivalis colonisation it is necessary to identify effective antigens that are involoved in virulence that have utility as immunogens to generate neutralising antibodies.
• P. gingivalis This complex was biochemically characterised and disclosed in International Patent Application No. PCT/AU96/00673, the disclosure of which is incorporated herein by reference.
• the complex consists of a 160 kDa Arg-specific proteinase with C-terminal adhesin domains (designated PrtR) associated with a 163 kDa Lys-specific proteinase also with C-terminal adhesin domains (designated PrtK).
• PrtR 160 kDa Arg-specific proteinase with C-terminal adhesin domains
• PrtK C-terminal adhesin domains
• the C-terminal adhesin domains of the PrtR and PrtK have homology with an haemagglutinin from P. gingivalis designated HagA.
• the gene encoding HagA has been disclosed in the international patent WO96/17936, the disclosure of which is incorporated herein by reference.
• the present inventors have identified a number of structurally and functionally significant sequences from the 300 kDa multiprotein complex of cysteine proteinases and adhesins which is a major virulence factor for P. gingivalis. These sequences are set OLit in Table 1. Table 1. Amino acid sequences of the PrtR-PrtK proteinase-adhesin complex of functional significance.
• the present invention consists in a composition for use in raising an immune response against Porphyromonas gingivalis, the composition including a suitable adjuvant and/or acceptable carrier or excipient and at least one peptide selected from the group consisting of:- FNGGISLANYTGHGSETAWGT;
• PNGTPNPNPNPNPGTTLSESF PNGTPNPNPNPNPNPGTTTLSESF;
• PAEWTTIDADGDGQG PAEWTTIDADGDGQG ;
• NSTQFNPVQNLTAEQAPNS EGSNEFAHVQNLTGSAVGQK;
• NPTQFNPVQNLTAEQAPNS NPTQFNPVQNLTAEQAPNS
• GNHEYCVEVKYTAGVSPKVCKDVTV GNHEYCVEVKYTAGVSPKKCVNVTV;
• GQYNYCVEVKYTAGVSPKVCKDVTV GQYNYCVEVKYTAGVSPKVCKDVTV
• GNHEYCVEVKYTAGVSPEVCVNVTV GQYNYCVEVKYTAGVSPEVCVNVTV
• the composition includes at least one peptide selected from the group consisting of:-
• composition includes more than one peptide the peptides may be present in the composition as individual peptides or in multimeric forms. Where multimeric forms are used the mul timer may comprise multiple copies of the same peptide, however, it is preferred that the mul timer includes differing peptides.
• Peptides (PASl and PAS2) of Table 1 represent sequences of the Arg- specific (PrtR45) and Lys-specific (PrtK48) cysteine proteinases which form the active site containing the Cys-His catalytic dyad.
• the remaining peptides (ABM peptides) represent adhesin binding motifs of the PrtR-PrtK protein-adhesin complex and HagA and together with the proteinase active site sequences, have proven to be effective as synthetic peptide vaccines.
• the present invention consists in a peptide, the peptide being selected from the group consisting of:-
• VNSTQFNPVKNLKAQPDGGDWLKWEAPSAK GEPSPYQPVSNLTATTQGQKVTLKWEAPSAK;
• PASWKTIDADGDGNN PASWKTIDADGDGNNW;
• GNHEYCVEVKYTAGVSPKKCVNVTV GNHEYCVEVKYTAGVSPKVCVNVTI;
• the peptide is selected from the group consisting of:-
• VTLKWDAPNGTPNPNPNPNPNPGTTTLSESF WIERTVDLPAGTKYVAFRHY
• PAEWTTIDADGDGQGW PAEWTTIDADGDGQGW
• EGSNEFAPVQNLTGSAVGQK EGSNEFAPVQNLTGSAVGQK .
• these peptides may be used as antigens in diagnostic tests or as immunogens in formulations.
• the present invention consists in an antibody preparation comprising antibodies specifically directed against the composition of the first aspect of the invention or the peptides of the second aspect of the invention.
• the antibodies may be either polyclonal or monoclonal antibodies.
• the present invention consists in a method of treating a subject suffering from Porphyromonas gingivalis infection, the method comprising administering to the subject an effective amount of the antibody preparation of the third aspect.
• the antibody preparation is administered as a mouth wash or as a dentifrice.
• the present invention consists in a method of reducing the prospect of P. gingivalis infection in an individual and/or severity of disease, the method comprising administering to the individual an amount of the composition of the first aspect effective to induce an immune response in the individual directed against P. gingivalis.
• Peptides can be synthesized using one of the several methods of peptide synthesis known in the art including standard solid phase peptide synthesis using t-butyloxycarbonyl amino acids (Mitchell et al., 1978, J. Org.
• a variety of methods for the synthesis of multivalent/multipeptide high molecular weight peptide molecules can be used to synthesize the peptide antigens. This will be achieved using known in the art and novel ligation strategies.
• Peptides from Table 1 can be synthesized in such away as to contain two ligands, which can be the same or different, which may or may not be the complementary ligand. These bi-modal peptides can incorporate any 9 / 1
• peptides may be used as antigens in diagnostic tests or as immunogens in formulations.
• the present invention consists in an antibody preparation comprising antibodies specifically directed against the composition of the first aspect of the invention or the peptides of the second aspect of the invention.
• the antibodies may be either polyclonal or monoclonal antibodies.
• the present invention consists in a method of treating a subject suffering from Porphyromonas gingivalis infection, the method comprising administering to the subject an effective amount of the antibody preparation of the third aspect.
• the antibody preparation is administered as a mouth wash or as a dentifrice.
• the present invention consists in a method of treating a subject suffering from Porphyromonas gingivalis infection, the method comprising administering to the subject an effective amount of a composition of the first aspect of the invention or a peptide of the second aspect of the invention.
• the composition or peptide is administered as a mouth wash or as a dentifrice.
• the present invention consists in a method of reducing the prospect of P. gingivalis infection in an individual and/or severity of disease, the method comprising administering to the individual an amount of the composition of the first aspect effective to induce an immune response in the individual directed against P. Gingivalis.
• Peptides can be synthesized using one of the several methods of peptide synthesis known in the art including standard solid phase peptide synthesis using t-butyloxycarbonyl amino acids (Mitchell et al.. 1978. J. Org. Chem. 43:2845-2852) using 9-fluorenylmethyloxycarbonyl (Fmoc) amino acids on a polyamide support (Druland et al., 1986, J. Chem. Soc.Perkin 10
• a variety of methods for the synthesis of multivalent/multipeptide high molecular weight peptide molecules can be used to synthesize the peptide antigens. This will be achieved using known in the art and novel ligation strategies.
• Peptides from Table 1 can be synthesized in such away as to contain two ligands, which can be the same or different, which may or may not be the complementary ligand. These bi-modal peptides can incorporate any ligand thus linkages such as thioether, thioester, hydrazone, oxime, thiazolidine can be utilised for the synthesis of multipeptide constructs Shao and Tarn., 1995, J. Am. Chem. Soc. 117, 3893-3899, Rose, et al 1996,
• ligand groups to form bi-modal peptides can be achieved by coupling a ligand on to free amino groups, which is known in the art. at the N- or C- terminus of a peptide or within the peptide sequence. This can be achieved by coupling eg. Fmoc(Fmoc) 2,3 diamino propionic acid or Fmoc Lys (Fmoc)-OH or orthogonally protected lysine residues such as Fmoc Lys (Mtt)-OH using standard peptide coupling protocols on to the N- terminus or introduced at the C-terminus or within the peptide sequence. After deprotection, ligand groups can be coupled on to the amino groups and 11
• Peptide ligation can be achieved in solution or on the solid phase.
• the incorporation of different ligands and selective protection of one ligand can allow the synthesis of multivalent, multipeptide constructs, where by, peptides are ligated sequentially.
• This strategy has the advantage that the orientation and order of peptides ligated is known and can be controlled.
• Protecting groups for ligands can be for example Fmoc, allyloxycarbonyl (Aloe) or nitrocinnamyloxycarbonyl (Noc) which are stable to standard cleavage conditions but are easily removed under basic conditions or catalytic allyl transfer.
• Figure 2 shows the ligation scheme for the synthesis of multivalent peptide constructs using bi-modal peptides.
• the protocol can be adapted for a variety of ligation chemistries by simply altering the ligands which are coupled to the peptide to form the bi-modal peptide.
• each peptide can be achieved on the solid phase. This can be achieved by synthesising a peptide on to the solid support via a base labile handle eg. 4-hydroxymethyl benzoic acid. This can allow full side chain deprotection of the peptide with the peptide remaining attached to the solid support. This would allow ligation to still be carried out in aqueous solvents similar to those used for solution phase ligation except that separation of the ligand product from unreacted bi-modal peptide can be achieved by simply washing the solid support. The reaction can be monitored by ninhydrin or trinitrobenzene sulphonic acid tests, where by, lysine residues within the bi-modal peptide would need to be protected eg.
• a base labile handle eg. 4-hydroxymethyl benzoic acid.
• Bi-modal peptides can be synthesized so that ligands are at the N- and C- terminus. This would allow the preparation of cyclic peptides and the formation of di-peptide constructs where by peptides can run parallel or anti parallel to each other by either coupling N- to N- and C- to C- termini or N- to C- termini together respectively (Figure 4). 12
• Another technique for the synthesis of multivalent peptide constructs is to ligate peptides on to an oligolysine support (Rose, et al 1996, Bioconjugate Chem. 7(5):552-556, Canne., et al 1995, J. Am. Chem. Soc. 117:2998-3007 and Lu., et al, 1991, Mol. Immunol 28(6):623-630).
• oligolysine support By incorporating a number of different ligands and or protected ligands on to the lysine support, peptides can be ligated to a particular position on the support.
• Ligation chemistries such as oxime or hydrazone with haloacylation and Friedal-Craft alkylation can be used sequentially without the need for ligand protection.
• Ligand protection can be used to increase the number of different peptides incorporated on to the lysine support.
• Figure 5 demonstrates the synthesis protocol.
• the final peptide construct may or may not contain all, sum or part of the peptides listed in Table 1. Also the construct may or may not contain promiscuous T-cell epitopes known in the art (Kaumaya et al 1994, in Solid Phase Synthesis, Ed Epton, R) or a derived sequence from structural/binding motifs of MHC class II binding peptides (O'Sullivan et al., 1991, J. Immunol, 147:2663-2669, Hammer et al, 1993, Cell, 74:197-203 and Alexander et al, 1994, Immunity, 1:751-761). Furthermore, lipid moieties such as palmitic acid or cholesterol can be included to enhance the immunogenic properties of the peptide construct.
• Enzymatic cleavable sequences known in the art can also be incorporated with the peptide construct.
• the synthetic peptide antigens identified in Table 1 are of particular interest for diagnostics and neutralisation by passive immunity through oral compositions containing neutralising antibodies and by vaccine development.
• the superiority of these synthetic peptide antigens to prior disclosed P. gingivalis antigens is that these sequences are homologous to structurally and functionally significant areas of the major P. gingivalis virulence factor the PrtR-PrtK proteinase-adhesin complex.
• Antibodies against the antigens can be used in oral compositions such as toothpaste and mouthwash to neutralise the antigens and thus prevent disease. Antigen-specific antibodies can also be used for the early detection of P. gingivalis in subgingival plaque samples by a diagnostic assay. A vaccine based on these antigens and suitable adjuvant delivered by nasal spray, orally or by injection to produce a specific immune response against these antigens thereby reducing colonisation and virulence of P. gingivalis and thereby preventing disease.
• the peptide antigens of the present invention may be used as immunogens in prophylactic and/or therapeutic vaccine formulations; or as an antigen in diagnostic immunoassays directed to detection of P.
• the synthetic peptides of the present invention may be used to generate antigen-specific antibody which may be useful for passive immunization and as reagents for diagnostic assays directed to detecting the presence of P. gingivalis in clinical specimens such as subgingival plaque samples.
• the synthetic peptide antigens described herein are safe and effective antigens for the preparation of a vaccine for the prevention of P. gingivalis- associated periodontal disease.
• FIG. 1 Synthesis of Bi-modal Peptides Although a specific example is shown here any ligand can be introduced at the a or e amino groups of lysine.
• acylation e.g. amino acid:HOBt:HBTU:DIPEA 1:1:1:1.5 in dimethyl formamide (DMF).
• Fmoc deprotection e.g. 20% piperidine in DMF.
• Levulinic acid diisopropyl carbodiimide (DIC) 2:1 in dichlorome thane (DCM). lh.
• DIC diisopropyl carbodiimide
• DCM dichlorome thane
• Mtt removal 3x 1% TFA in DCM, 3 mins.
• Fmoc-Hydrazino benzoic acid DIC 2:1, in DCM, lh.
• Acid cleavage e.g. TFA : water 95:5. 14
• Figure 2 Synthesis of multivalent peptide constructs using bi-modal peptides.
• Different ligation chemistries can be used by synthesising peptides with different ligands and synthesising non- complementary ligands on to the same peptide, thereby avoiding proected ligands.
• the square symbol indicates protection, (L) ligand, (P) peptide.
• Figure 3 Synthesis of multivalent peptide constructs using bi-modal peptides by solid phase, (a) Deprotection and ligation. The S-acetyl protecting group is removed by aqueous hydroxyamine 0.05 M, pH 7.3. After washing the first peptide can be ligated on to the SH group, 6 M aqueous gua idine hydrochloride and 0.05 M EDTA pH 6.4-6.5 adjusted by 1 M Tris.HCl under nitrogen. Ligation buffer can contain organic solvents such as acetonitrile. (b) Deprotection, the S-acetyl protecting group can be removed by aqueous hydroxyamine 0.05 M, pH 7.3.
• Figure 4 Cyclization using bi-modal peptides.
• the peptides shown are from Table 1 and present the active site peptides from prtR 45.
• Ligation conditions can be varied to included scavengers commonly used in peptide synthesis and different acidic conditions to enhance the Friedal-Craft alkylation.
• the S-acetyl protecting group can removed by aqueous 15
• FIG. 5 Synthesis of multivalent multiple antigenic peptides (MAPs) using alternate ligation chemistries. By using different ligation strategies a vareity of peptides can be ligated onto a single multiple antigenic peptide. The example shown is of peptides listed in Table 1. (a) Ligation, 95% aqueous
• Ligation can be monitored by reverse phase analytical HPLC and mass spectrometry. Deprotection, Aloe can removed by palladium(0)-catalyzed allyl group transfer to a basic receptor, after purifaction the second peptide can be ligated on to the MAP, (c) 8 M urea and 0.1 M NaH 2 PO 4 (pH range 3- 4.7).
• FIG. 6 Gel Filtration FPLC of pooled and concentrated fractions eluting from Q sepharose anion exchange FPLC.
• Anion exchange fractions eluting between 160-246 mM NaCl and representing the leading edge of the main peak of proteolytic/amidolytic activity were pooled, equilibrated in TC buffer pH 7.4 containing 50 mM NaCl, concentrated and applied to Superose 12 HR 10/30 gel filtration column using the same buffer at a flow rate of 0.3 ml min "1 .
• Fractions (0.5 ml) were assayed for proteolytic/amidolytic activity using azocasein, Bz-L-Arg-pNA and z-L-Lys-pNA.
• Amidolytic activity of each 0.5 ml fraction with Bz-L-pNA is shown by the histogram.
• FIG. 7 SDS-PAGE (bolied/reduced conditions) of the anion exchange (Mono Q) peak eluting at 200 mM NaCl and containing only Arg-specific activity.
• Lane 1 Pharmacia low molecular mass standards;
• lane 2 Purified 50 kDa Arg-specific proteinase, PrtRIl50.
• FIG. 8 An alignment of the deduced amino acid sequences of PrtRIl50, PrtR45 Arg-specific proteinase and PrtK48 Lys-specific proteinase with optimised similarity.
• the amino acyl residues of PrtRIl50 are numbered from the N-terminal residue of the mature protein. * indicates an identical residue 16
• FIG. 10 Competitive Binding Assay demonstrating binding of the TLCK- inactivated PrtR-PrtK proteinase-adhesion complex to the synthetic peptide corresponding to the putative adhesin binding motif (ABM). -•-•- ABM synthetic peptide.
• PYQPVSNLTATTQGQKNTLKWDAPSTK - ⁇ - ⁇ - Control peptide, FNGGISLANYTGHGSETAWGT corresponding to residues 428-448 of PrtR45. - - ⁇ - casein. See Materials and Methods for details.
• FIG. 11 Average lesion size of mice challenged with Porphyromonas gingivalis in a mouse abcsess model.
• BALB/c mice (6 per group) were inoculated (s.c.) with 50 ⁇ g of antigen emulsified in CFA and IFA for the primary and secondary inoculations and then challenged (s.c.) with 8 X 10 9 cells of P. gingivalis strain 33277.
• This invention relates to an oral composition and an immunogenic composition for the suppression of the pathogenic effects of the intra-oral bacterium Porphyromonas gingivalis associated with periodontal disease. It also relates to diagnostic tests for the presence of Porphyromonas gingivalis in subgingival plaque samples and specific anti-P. gingivalis antibodies in sera.
• the peptide antigens of Table 1 can be synthesized individually or as multimetric or multipeptide constructs.
• the synthetic peptide antigens are used to generate polyclonal or monoclonal antibodies using standard techniques.
• the animals used for antibody generation can be mice, rabbits, goats, chickens, sheep, horses, cows etc.
• a high antibody titre against the antigens is detected by immunoassay the animals are bled or eggs or milk are collected and the serum prepared and/or antibody purified using standard techniques or monoclonal antibodies produced by fusing spleen cells with myeloma cells using standard techniques.
• the antibody immunoglobulin fraction
• This invention relates to an oral composition and an immunogenic composition for the suppression of the pathogenic effects of the intra-oral bacterium Porphyromonas gingivalis associated with periodontal disease. It also relates to diagnostic tests for the presence of Porphyromonas gingivalis in subgingival plaque samples and specific anti-P. gingivalis antibodies in sera.
• the peptide antigens of Table 1 can be synthesized individually or as multimetric or multipeptide constructs.
• the synthetic peptide antigens are used to generate polyclonal or monoclonal antibodies using standard techniques.
• the animals used for antibody generation can be mice, rabbits, goats, chickens, sheep, horses, cows etc.
• a high antibody titre against the antigens is detected by immunoassay the animals are bled or eggs or milk are collected and the serum prepared and/or antibody purified using standard techniques or monoclonal antibodies produced by fusing spleen cells with myeloma cells using standard techniques.
• the antibody immunoglobtuin fraction
• the antiserum or the milk is saturated with ammonium sulphate to produce a precipitate, followed by dialyzing the precipitate against physiological saline to obtain the purified immunoglobulin fraction with the specific antibody.
• the preferred antibody is obtained from the equine antiserum and the bovine antiserum and milk.
• the antibody contained in the antiserum and milk obtained by immunising the animal with the antigens is blended into the oral composition.
• the antiserum and milk as well as the antibody separated and purified from the antiserum and milk may be used. Each of these materials may be used alone or in combination of two or more.
• Antibodies can be used in oral compositions such as toothpaste and mouthwash to neutralise P. gingivalis and thus prevent disease.
• the antibodies can also be used for the early detection of P. gingivalis in subgingival plaque samples by a chairside Enzyme Linked Immunosorbent Assay (ELISA).
• the amount of the above antibodies administered is 0.0001 -50 g/kg/day and that the content of the above antibodies is 0.0002 - 10% by weight preferably 0.002 -5% by weight of the composition.
• the oral composition of this invention which contains the 18
• serum or milk antibody may be prepared and used in various forms applicable to the mouth such as dentifrice including toothpastes, toothpowders and liquid dentifrices, mouthwashes, troches, chewing gums, dental pastes, gingival massage creams, gargle tablets, dairy products and other foodstuffs.
• the oral composition according to this invention may further include additional well known ingredients depending on the type and form of a particular oral composition.
• the oral composition may be substantially liquid in character, such as a mouthwash or rinse.
• the vehicle is typically a water-alcohol mixture desirably including a humectant as described below.
• the weight ratio of water to alcohol is in the range of from about 1:1 to about 20:1.
• the total amount of water-alcohol mixture in this type of preparation is typically in the range of from about 70 to about 99.9% by weight of the preparation.
• the alcohol is typically ethanol or isopropanol. Ethanol is preferred.
• the pH of such liquid and other preparations of the invention is generally in the range of from about 4.5 to about 9 and typically from about 5.5 to 8.
• the pH is preferably in the range of from about 6 to about 8.0, preferably 7.4.
• the pH can be controlled with acid (e.g. citric acid or benzoic acid) or base (e.g. sodium hydroxide) or buffered (as with sodium citrate, benzoate. carbonate, or bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc).
• the oral composition may be substantially solid or pasty in character, such as toothpowder, a dental tablet or a dentifrice, that is a toothpaste (dental cream) or gel dentifrice.
• the vehicle of such solid or pasty oral preparations generally contains dentally acceptable polishing material.
• polishing materials are water- insoluable sodium metaphosphate, potassium metaphosphate, tricalcium phosphate, dihydrated calcium phosphate, anhydrous diealeium phosphate, calcium pyrophosphate, magnesium orthophosphate, trimagnesium phosphate, calcium carbonate, hydrated alumina, calcined alumina, aluminum silicate, zirconium silicate, silica, bentonite. and mixtures thereof.
• Other suitable polishing material include the particulars thermosetting resins such as melamine-. phenolic, and urea-formaldehydes, and cross-linked polyepoxides and polyesters.
• Preferred polishing materials include crystalline 19
• silica having particle sized of up to about 5 microns, a mean particle size of up to about 1.1 microns, and a surface area of up to about 50,000 cm 2 /gm., silica gel or colloidal silica, and complex amorphous alkali metal aluminosilicate.
• a polishing agent of colloidal silica such as those sold under the trademark SYLOID as Syloid 72 and Syloid 74 or under the trademark SANTOCEL as Santocel 100
• alkali metal alumino-silicate complexes are particularly useful since they have refractive indices close to the refractive indices of gelling agent-liquid (including water and/or humectant) systems commonly used in dentifrices.
• insoluble sodium metaphosphate may be formed in any suitable manner as illustrated by Thorpe's Dictionary of Applied Chemistry, Volume 9, 4th Edition, pp. 510-511.
• Madrell's salt and Kurrol's salt are further examples of suitable materials.
• These metaphosphate salts exhibit only a minute solubility in water, and therefore are commonly referred to as insoluble metaphosphates (IMP).
• IMP insoluble metaphosphates
• impurities usually a few percent such as up to 4% by weight.
• the amount of soluble phosphate material which is believed to include a soluble sodium trimetaphosphate in the case of insoluble metaphosphate, may be reduced or eliminated by washing with water if desired.
• the insoluble alkali metal metaphosphate is typically employed in powder form of a particle size such that no more than 1% of the material is larger than 37 microns.
• the polishing material is generally present in the solid or pasty compositions in weight concentrations of about 10% to about 99%. Preferably, it is present in amounts from about 10% to about 75% in toothpaste, and from about 70% to about 99% in toothpowder. In toothpastes, when the polishing material is silicious in nature, it is generally present in amount of about 10-30% by weight. Other polishing materials are typically present in amount of about 30-75% by weight.
• the liquid vehicle may comprise water and humectant typically in an amount ranging from about 10% to about 80% by weight of the preparation.
• humectant typically in an amount ranging from about 10% to about 80% by weight of the preparation.
• Glycerine, propylene glycol, sorbitol and polypropylene glycol exemplify suitable humectants/carriers. Also 20
• Toothpaste, creams and gels typically contain a natural or synthetic thickener or gelling agent in proportions of about 0.1 to about 10, preferably about 0.5 to about 5% w/w.
• a suitable thickener is synthetic hectorite, a synthetic colloidal magnesium alkali metal silicate complex clay available for example as Laponite (e.g. CP, SP 2002, D) marketed by Laporte Industries Limited.
• Laponite D is, approximately by weight 58.00% Si0 2 , 25.40% MgO, 3.05% Na 2 O, 0.98% Li 2 O, and some water and trace metals. Its true specific gravity is 2.53 and it has an apparent bulk density of 1.0 g/ml at 8% moisture.
• thickeners include Irish moss, iota carrageenan, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose (e.g. available as Natrosol), sodium carboxymethyl cellulose, and colloidal silica such as finely ground Syloid (e.g. 244).
• Irish moss iota carrageenan
• gum tragacanth starch
• polyvinylpyrrolidone hydroxyethylpropylcellulose
• hydroxybutyl methyl cellulose hydroxypropyl methyl cellulose
• sodium carboxymethyl cellulose hydroxyethyl cellulose
• colloidal silica such as finely ground Syloid (e.g. 244).
• Solubilizing agents may also be included such as humectant polyols such propylene glycol, dipropylene glycol and hexylene glycol, cellosolves such as methyl cellosolve and ethyl cellosolve, vegetable oils and waxes containing at least about 12 carbons in a straight chain such as olive oil, castor oil and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate.
• humectant polyols such propylene glycol, dipropylene glycol and hexylene glycol
• cellosolves such as methyl cellosolve and ethyl cellosolve
• vegetable oils and waxes containing at least about 12 carbons in a straight chain such as olive oil, castor oil and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate.
• a jar of mouthrinse will have a label describing it, in substance, as a mouthrinse or mouth wash and having directions for its use; and a toothpaste, cream or gel will usually be in a collapsible tube, typically aluminium, lined lead or plastic, or other squeeze, pump or pressurized dispenser for metering out the contents, having a label describing it, in substance, as a toothpaste, gel or dental cream.
• Organic surface-active agents are used in the compositions of the present invention to achieve increased prophylactic action, assist in achieving thorough and complete dispersion of the active agent throughout the oral cavity, and render the instant compositions more cosmetically acceptable.
• the organic surface-active material is preferably anionic, 21
• anionic surfactants are water-soluble salts of higher fatty acid monoglyceride monosulfates, such as the sodium salt of the monosulfated monoglyceride of hydrogenated coconut oil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate, alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higher alkylsulfo-acetates, higher fatty acid esters of 1,2-dihydroxy propane sulfonate, and the substantially saturated higher aliphatic acyl amides of lower aliphatic amino carboxylic acid compounds, such as those having 12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and
• Examples of the last mentioned amides are N-lauroyl sarcosine, and the sodium, potassium, and ethanolamine salts of N-lauroyl, N-myristoyl, or N- palmitoyl sarcosine which should be substantially free from soap or similar higher fatty acid material.
• the use of these sarconite compounds in the oral compositions of the present invention is particularly advantageous since these materials exhibit a prolonged marked effect in the inhibition of acid formation in the oral cavity due to carbohydrates breakdown in addition to exerting some reduction in the solubility of tooth enamel in acid solutions.
• Examples of water-soluble nonionic surfactants suitable for use with antibodies are condensation products of ethylene oxide with various reactive hydrogen-containing compounds reactive therewith having long hydrophobic chains (e.g.
• condensation products contain hydrophilic polyoxyethylene moieties, such as condensation products of poly (ethylene oxide) with fatty acids, fatty alcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate) and polypropyleneoxide (e.g. Pluronic materials).
• Surface active agent is typically present in amount of about 0.1-5% by weight. It is noteworthy, that the surface active agent may assist in the dissolving of the antibody of the invention and thereby diminish the amount of solubilizing humectant needed.
• whitening agents such as whitening agents, preservatives, silicones, chlorophyll compounds and/or ammoniated material such as urea, diammonium phosphate, and mixtures thereof.
• silicones such as silicones, chlorophyll compounds and/or ammoniated material such as urea, diammonium phosphate, and mixtures thereof.
• ammoniated material such as urea, diammonium phosphate, and mixtures thereof.
• flavoring or sweetening material may also be employed.
• suitable flavoring constituents are flavoring oils, e.g. oil of spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon, and orange, and methyl salicylate.
• suitable sweetening agents include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP (aspartyl phenyl alanine, methyl ester), saccharine, and the like.
• flavor and sweetening agents may each or together comprise from about 0.1% to 5% more of the preparation.
• an oral composition according to this invention such as mouthwash or dentifrice containing the composition of the present invention is preferably applied regularly to the gums and teeth, such as every day or every second or third day or preferably from 1 to 3 times daily, at a pH of about 4.5 to about 9, generally about 5.5 to about 8, preferably about 6 to 8, for at least 2 weeks up to 8 weeks or more up to a lifetime.
• compositions of this invention can be incorporated in lozenges, or in chewing gum or other products, e.g. by stirring into a warm gum base or coating the outer surface of a gum base, illustrative of which may be mentioned jelutong, rubber latex, vinylite resins, etc., desirably with conventional plasticizers or softeners, sugar or other sweeteners or such as glucose, sorbitol and the like.
• Another important form of the invention is a immunogenic composition based on the synthetic peptide antigens and suitable adjuvant delivered by nasal spray, orally or by injection to produce a specific immune response against the antigen thereby reducing colonisation of P. gingivalis and reducing virulence thereby preventing disease.
• the peptide antigens described herin are safe and effective antigens for the preparation of a vaccine for the prevention of P. gi-ngiv ⁇ /is-associated periodontal disease.
• antigenic peptide produced may be used to generate P. gingivalis antisera useful for passive immunization against periodontal disease and infections caused by P. gingivalis. 23
• HBTU O-Benzotriazole-N,iV,N',iV-tetramethyluronium hexafluorophosphate
• HOBt lhydroxybenzotriazole
• DIPEA diisopropylethylamine
• DMF N,N-dimethylformamide
• piperidine trifluoroacetic acid
• Fmoc 9- fluorenylmethoxycarbonyl
• DBU 1.8-diazabicyclo[5.4.0]undec-7-ene
• P. gingivalis W50 was grown in batch culture (5 L) and harvested at late logarithmic phase by centrifugation (5,000 x g, 20 min. 4 °C). Cells were washed once with 150 ml TC buffer (20 mM Tris-HCl pH 7.4 and 5 mM
• the sonicate was centrifuged (100,000 x g, 30 min, 4 °C) and the supernatant filtered (0.22 ⁇ m) prior to anion-exchange FPLC.
• the sonicate was applied to an anion-exchange column (Hiload XK 16/10 Q Sepharose, Pharmacia-LKB) cooled to 4 °C, in multiple injections using a 50 ml superloop (Pharmacia-LKB).
• the sonicate was eluted using a linear gradient from 0-100% buffer B over 90 min at a flow rate of 2.0 ml min "1 .
• r values of eluant peaks were determined using gel filtration molecular mass standards (Pharmacia-LKB).
• the peak eluting at 50 kDa containing only Arg-specific amidolytic activity was washed in TC buffer containing 50 mM NaCl using a centricon-10 concentrator (Amicon) and applied to a Mono Q (HR 5/5) anion- exchange column using a 5 ml loop and eluted using a linear gradient of 0- 100% buffer B at a flow rate 1.0 ml min "1 .
• Buffer A was TC buffer containing 150 mM NaCl
• buffer B was TC buffer containing 500 mM NaCl.
• Absorbance was monitored at 280 nm and fractions collected at 4 °C using a Frac 100 fraction collector.
• the P. gingivalis W50 LambdaGEM ® -12 genomic library described previously (Slakeski et al., 1996) was screened using synthetic oligonucleotides derived from the nucleotide sequence of prtR (Slakeski et al., 1996) corresponding to the N-terminal sequence of PrtR45.
• Oligonucleotide probes were 5' end-labelled using ⁇ 32 P ATP and T4 polynucleotide kinase. Approximately 1.5 x 10 4 phage were screened by lifting onto Nylon membrane filters and hybridising with radiolabelled oligonucleotides overnight in hybridisation buffer: 6 x SSC (SSC is 15 mM sodium citrate, 150 mM NaCl pH 8.0), 0.25% SDS, 5 x Denhardts solution (Sambrook et al.. 1989) and 100 ⁇ g/ml salmon sperm DNA at 49 °C. Filters were washed extensively in a solution of 2 x SSC containing 0.1% SDS (w/v) at 49 °C. Phage from positively-hybridising plaques were purified using standard procedures (Sambrook et al., 1989). Phage DNA was digested with Eco72 I and the resulting fragments ligated into Sma I-BAP pUCl ⁇
• the PCR was carried out in a final volume of 100 ⁇ l and each reaction mixture contained 100 ng P. gingivalis W50 genomic DNA, 0.2 mM dNTPs, 1.5 mM MgCl 2 , 100 pmol of each primer. 20 mM Tris-HCl, pH 8.4, 50 mM KC1 and 2.5 U Taq DNA Polymerase (Gibco BRL).
• the reaction mixture was heated at 95 °C for 3 min and then subject to 25 cycles of DNA denaturation at 95 °C for 30 s, primer annealing at 40 °C for 1 min and extension at 72 °C for 2 min. Following cycling, the reaction mixture was finally heated at 72 °C for 5 min. 26
• Amplified DNA was purified using a PCR Spinclean Kit (Progen) and sequenced across the Eco 721 site in both directions.
• the high molecular mass, cell-associated proteinase-adhesin complexes (PrtR-PrtK complexes) of P. gingivalis W50 were purified using a combination of anion-exchange, gel filtration and Arg-sepharose affinity chromatography from a cell sonicate as described previously (Bhogal et al., 1997).
• the complexes were characterised using SDS-PAGE, transblotting and sequence analysis and assayed for enzymic activity using Bz-L-Arg-pNA and Z-L-Lys-pNA substrates (Bhogal et al., 1997).
• Solid-phase peptide synthesis Peptides ⁇ vere synthesised manually using standard Fmoc solid-phase peptide synthesis protocols.
• the peptides were assembled as the carboxyamide form using Fmoc-Pal-Peg-PS resin (PerSeptive Biosystems Inc., Framingham, MA). Coupling was accomplished with HBTU/HOBt activation using 4 equiv of Fmoc-amino acid and 6 equiv of DIPEA.
• the Fmoc group was removed by 2% v/v DBU in DMF containing 2% v/v piperidine.
• a solution (1 mg/ml) of the PrtR-PrtK proteinase-adhesion complex (inactivated with lmJVI TLCK) was incubated with known concentrations of ABM peptide, control peptide and casein for 1 hour and then transferred to the microtitre plates coated with the ABM peptide. Following incubation for 2 hours at 37 °C the plates were washed (5 x PBST). A 1/10,000 dilution of rabbit anti -PrtR-PrtK antisera in PBST containing 1% w/v skim milk powder was then added to the washed wells and incubated for 2 hours at 37 °C.
• Bound antibody was detected by incubation with horseradish peroxidase-conjugated goat immunoglobulin (Ig) directed against rabbit Ig (BioRad, Richmond, CA) for 1.5 hours at 37 °C. After washing (5 x PBST), substrate (0.4mM 3,3',5,5'-tetramethylbenzidine in 0.1M sodium acetate/citric acid buffer containing 0.004% v/v hydrogen peroxide) was added and colour development was stopped by addition of 2M H 2 SO 4 . Optical density (O.D.) at 450 nm was measured using a BioRad microplate reader model 450.
• the P. gingivalis W50 cell sonicate contained 0.36 mg ml "1 protein and 2.4 and 1.1 ⁇ mol min " 1 mg protein "1 activity with 1.0 mM Bz-L-Arg-pNA and z-L-Lys-pNA as substrates respectively at 25 °C.
• the sonicate was subjected to Q-sepharose anion exchange FPLC and proteolytic/amidolytic activity eluting between 160-246 mM NaCl was collected and concentrated using a centripep and centricon-10 concentrator (Amicon, Sydney, Australia). 28
• This fraction represented the leading edge of the main peak of proteolytic/amidolytic activity and contained the highest ratio of Arg-specific activity to Lys-specific activity.
• the fraction was applied to a Superose 12 gel filtration column (Fig. 6).
• Arg- and Lys- specific activity was associated with the high molecular mass eluting material corresponding to peaks with M r values of 0.6 - 2.0 x 10 6 Da and 300 kDa as reported previously (Bhogal et al., 1997).
• M r values 0.6 - 2.0 x 10 6 Da and 300 kDa
• a lower molecular mass peak of 50 kDa was also observed, which displayed only Arg-specific activity and this peak was collected for further purification.
• the 50 kDa gel filtration peak was applied to a Mono Q anion exchange column and upon application of a NaCl gradient the Arg-specific activity eluted in a distinct peak at a NaCl concentration of 200 mM NaCl with a 28-fold purification over the original crude sonicate.
• the peak containing Arg-specific activity was subjected to SDS-PAGE which confirmed a single 50 kDa protein band (Fig. 7).
• the 50 kDa band was transblotted and subjected to N-terminal sequence analysis which provided the amino acyl sequence
• YTPVEEKENGRMIVIVPKKYEEDIED The specificity of the 50 kDa proteinase for arginyl residues was confirmed by the enzyme cleaving Bz-L- Arg-pNA but not z-L-Lys-joNA.
• the Arg-specific 50 kDa enzyme was activated by thiols (particularly cysteine), not inhibited by the serine proteinase inhibitors, phenylmethyl sulfonyl fluoride or 4-(2-aminoethyl)- benzenesulfonyl fluoride but inhibited by sulphydryl-directed reagents, leupeptin and EDTA at similar concentrations to that which inhibited the PrtR45 (Bhogal et al., 1997). Inhibition with EDTA could be reversed by the addition of excess Ca 2+ and the pH optimum of the enzyme was 8.0 with minimal activity below pH 6.0.
• the prtRII OKF comprises 2208 bp (736 a.a. residues) and encodes a preproprotein consisting of a putative leader sequence and a profragment followed by the mature Arg-specific proteinase (507 a.a. residues) containing the exact N-terminal amino acyl sequence obtained for the purified 50 kDa enzyme (PrtRII ⁇ O).
• the N-terminal sequence of the mature protein like PrtR45, is immediately preceded by an Arg residue in the profragment.
• the prtRII gene exhibits a high degree of similarity with the 5' two fifths of the prtR gene which encodes PrtR45 and its associated adhesins (Figs. 8 & 9).
• a comparison of the two translated sequences shows an overall similarity of 76% and 80% for the translated preprofragment and the proteinase domain, respectively.
• the prtRII does not encode any of the C-terminal haemagglutinin/adhesin domains encoded by the prtR and prtK enes being consistent with the finding that the purified PrtRIl50 proteinase was not associated with adhesins.
• the M r of the PrtRII50 mature proteinase deduced from the translated prtRII gene sequence is 55.6 kDa which is consistent with the 50 kDa obtained by SDS-PAG ⁇ (Fig. 7) and is slightly larger than the deduced M r of 53.9 kDa for PrtR45 (Bhogal et al., 1997).
• TLCK-inactivated PrtR-PrtK complex to the ABM peptide was demonstrated in a competitive binding assay where a 5-100 fold molar excess of the ABM peptide in solution was required to inhibit binding of the complex to the ABM peptide adsorbed onto the microtitre plate (Fig. 10).
• a control peptide. FNGGISLANYTGHGSETAWGT corresponding to residues 428-448 of PrtR45. as well as casein did not inhibit the binding of the TLCK-inactivated PrtR- PrtK complex to the adsorbed ABM peptide.
• the anti-PrtR-PrtK antisera did not bind to the ABM peptide in the absence of the PrtR-PrtK complex.
• PrtRII ⁇ O is slightly larger than PrtR45 on SDS-PAGE (M r 50 kDa) and thirdly there are four amino acid substitutions in the first 25 N-terminal residues of PrtRIl50.
• PrtRII50 has a Glu at position 8 instead of Gin, a Pro at position 17 instead of Ala, a Glu at position 22 instead of Gly and a Glu at position 25 instead of the Lys in PrtR45 (Fig. 8).
• PrtRIl50 The substitutions at positions 17, 22 and 25 found in PrtRIl50, that enabled the gene product to be unequivocally differentiated by N-terminal sequence analysis from the mature PrtR45 proteinase of the prtR ⁇ rgpA), were not found in the rgpB.
• the differences in N-terminal sequence and size of the mature proteinases enabled the differentiation of the discrete 50 kDa Arg-specific proteinase (PrtRIl50) from the 45 kDa Arg-specific proteinase (PrtR45) found associated with adhesins.
• Solid-Phase Peptide Synthesis Peptides were synthesised manually or using a 431A ABI peptide synthesiser. Standard solid-phase peptide synthesis protocols for Fmoc chemistry were used throughout. Peptides were assembled as the carboxyamide form using Fmoc-Pal-Peg-PS resin (PerSeptive Biosystems Inc., Framingham, MA). Coupling was accomplished with HBTU/HOBt activation using 4 equiv of Fmoc-amino acid and 6 equiv of DIPEA. The Fmoc group was removed by 2% v/v DBU in DMF containing 2% v/v piperidine.
• the peptide precipitate was then dissolved in 5 to 10 mL of water containing 0.1% v/v TFA and insoluble residue removed by centrifugation.
• SAMA S-Acetylmercaptoacetic acid
• TNBSA trinitrobenzene sulphonic acid
• Chromatograms were developed using solvent A and solvent B at a flow rate of 1 mL/min and a 0- 100% linear gradient of solvent B formed over 30 min. Material eluted from the columns was detected by determining the absorbance at 214 nm. Peptide fractions were pooled and lyophilised. Peptides were analysed by mass spectrometry using a PerSeptive Biosystems Voyager DE MALDI-TOF.
• the peptide-DT conjugate was separated from unreacted peptide by gel filtration using a PDIO column equilibrated in PBS pH 7.4 and lyophilised. The reaction was monitored using the Ellmans test. The conjugation yields of SAMA-peptides to MBS-DT ranged from 34% to 45% indicating that 3 to 4 peptides were coupled per DT molecule.
• mice 6-8 weeks old were immunised subcutaneously with either 50 ⁇ g of the peptide-DT conjugate, 50 ⁇ g of DT or 2 x 10° formalin killed cells of Porphyromonas gingivalis strain 33277 emulsified in complete Freund's adjuvant (CFA). After 30 days the mice were injected subcutaneously with antigen (either 50 ⁇ g of the peptide-DT conjugate, 50 ⁇ g of DT or 2 x 10 9 formalin killed cells of Porphyromonas gingivalis strain
• mice were challenged with 8 x 10 9 cells of P. gingivalis (200 ⁇ L) by subcutaneous injection in the abdomen and weighed and lesion size measured over 10 days. Lesion sizes are expressed as mm 2 and were statistically analysed using a Kruskal-Wallis one-way ANOVA and Mann- Whitney U-Wilcoxon rank sum W test.
• Antisera against the PASl peptides inhibited both Arg- and Lys- specific proteolytic activity which therefore may explain the excellent protection conferred by immunisation with these peptides.
• the inhibition of proteolytic activity by the anti-PASl antisera suggests that these antibodies may have utility in a mouthwash, toothpaste or other intra-oral delivery vehicle to neutralise the P. gingivalis proteases and their damaging effects.
• antisera against the adhesin binding motifs, particularly ABMl are examples of adhesin binding motifs, particularly ABMl
• ABM2 and ABM3 may have utility in oral care products and pharmaceuticals to block adherence and therefore colonisation of P. gingivalis.
• peptides of Table 1 were synthesized using standard Fmoc or tBoc synthesis strategies and multipeptide constructs were synthesized using the strategies outlined in Figs. 1-5.
• Serum antibodies were obtained by immunising horses, rabbits, sheep or dairy cows.
• Immunizations were carried out using standard procedures. The initial immunisation was with a mixture of the antigen and Freund's incomplete adjuvant. The antibodies could be recovered from the animals serum or milk using standard procedures.
• the P. gingivalis peptide antigens described herein can be synthesized for use as immunogens in vaccine formulations; and as antigens for diagnostic assays or for generating P. gingiv ⁇ /is-specific antisera of therapeutic and/or diagnostic value.
• the peptides disclosed in Table 1 can be synthesized individually or chemically-linked using any one of a number of strategies well known in the art. Examples of some strategies which can be used are set out in Figs. 1 - 5.
• the peptides can be synthesized using one of the several methods of peptide synthesis known in the art including standard solid phase peptide synthesis using tertbutyloxycarbonyl amino acids (Mitchell et al., 1978, J. Org. Chem. 43:2845-2852), using 9-fluorenylmethyloxycarbonyl amino acids on a polyamide support (Dryland et al., 1986, J. Chem. So. PerMn Trans.
• amino acid sequences of the antigens described herein may be altered by replacing one or more amino acids with functionally equivalent amino acids resulting in an alteration which is silent in terms of an observed difference in the physicochemical behaviour of the peptide, or oligopeptide or chimera.
• Functionally equivalent amino acids are known in the art as amino acids which are related and/or have similar polarity or charge.
• an amino acid sequence which is substantially that of the amino acid sequences depicted in the Sequence Listing herein refers to an amino acid sequence that contains substitutions with functionally equivalent amino acids without changing the primary biological function of the peptide, oligopeptide or chimera.
• Purified synthetic peptides may be used as antigens in immunoassays for the detection of P. gingiv ⁇ iis-specific antisera present in the body fluid of an individual suspected of having an infection caused by P. gingivalis.
• the detection of antigens or related peptides in immunoassays includes any immunoassay known in the art including, but not limited to, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich” assay, precipitin reaction, agglutination assay, fluorescent immunoassay, and chemiluminescence-based immunoassay.
• This embodiment of the present invention is to provide peptide antigens of Table 1 to be used as immunogens in a prophylactic and/or therapeutic vaccine for active immunization to protect against or treat infections caused by P. gingivalis.
• an antigen of P. gingivalis comprising a synthetic peptide construct should be immunogenic, and induce functional antibodies directed to one or more surface-exposed epitopes on intact bacteria, wherein the epitope(s) are conserved amongst strains of P. gingivalis.
• the dipeptide PAS1-PAS2 construct (Fig. 4) having the properties desirable of a vaccine antigen, the dipeptide construct can be synthesized using the method described herein in Example 3. 40
• the synthetic peptide is included as the relevant immunogenic material in the vaccine formulation, and in therapeutically effective amounts, to induce an immune response.
• Many methods are known for the introduction of a vaccine formulation into the human or animal to be vaccinated. These include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, ocular, intranasal, and oral administration.
• the vaccine may further comprise a physiological carrier such as a solution, a polymer or liposomes; and an adjuvant, or a combination thereof.
• adjuvants are used in conjunction with vaccine formulations.
• the adjuvants aid by modulating the immune response and in attaining a more durable and higher level of immunity using smaller amounts of vaccine antigen or fewer doses than if the vaccine antigen were administered alone.
• adjuvants include incomplete Freund's adjuvant (ISA), Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostrearate), oil emulsions, Ribi adjuvant, the pluronic polyols, polyamines, Avridine, Quil A, saponin, MPL, QS-21, and mineral gels such as aluminum hydroxide, aluminum phosphate, etc.
• Another embodiment of this mode of the invention involves the production of antigen-specific amino acid sequences as a hapten, i.e. a molecule which cannot by itself elicit an immune response.
• the hapten may be covalently bound to a carrier or other immunogenic molecule which will confer immunogenicity to the coupled hapten when exposed to the immune system.
• a antigen-specific hapten linked to a carrier molecule may be the immunogen in a vaccine formulation.
• immunization may be passive, i.e. immunization comprising administration of purified immunoglobulin containing antibody against synthetic peptides.

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