US20100092471A1 - Porphyromonas Gingivalis Polypeptides Useful in the Prevention of Periodontal Disease - Google Patents

Porphyromonas Gingivalis Polypeptides Useful in the Prevention of Periodontal Disease Download PDF

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US20100092471A1
US20100092471A1 US12/306,495 US30649507A US2010092471A1 US 20100092471 A1 US20100092471 A1 US 20100092471A1 US 30649507 A US30649507 A US 30649507A US 2010092471 A1 US2010092471 A1 US 2010092471A1
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amino acid
acid sequence
seq
gingivalis
protein
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Stuart Geoffrey Dashper
Ching Seng Ang
Paul David Veith
Eric Charles Reynolds
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Oral Health Australia Pty Ltd
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Oral Health Australia Pty Ltd
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/18—Dental and oral disorders

Definitions

  • the present invention relates to compositions and methods of isolating Porphyromonas gingivalis ( P. gingivalis ) proteins useful in the prevention of and treatment of periodontal disease. More particularly, the invention is directed to vaccine compositions and methods based on P. gingivalis proteins identified to be regulated by haem availability that can be used in the prevention and treatment of periodontal disease.
  • P. gingivalis Porphyromonas gingivalis
  • Periodontal disease is a chronic bacterial infection that affects the gums and bone supporting the teeth. Periodontal disease begins when the bacteria in plaque (the sticky biofilm that constantly forms on teeth) causes the gums to become inflamed. Periodontal disease can affect the gingival tissue (gums); periodontal membrane (connective tissue embedded in the cementum and alveolar bone); cementum (mineralized connective tissue covering the roots of the teeth); and the alveolar bone (bone socket), Depending on the progression of the disease, there may occur a destruction of periodontal membranes, alveolar bone loss, and apical migration of the connective tissue attachment. Advanced periodontal disease may result in the formation of periodontal pockets harbouring bacterial plaque, and progressive loosening and eventual loss of teeth.
  • Periodontal disease includes gingivitis that can advance to periodontitis.
  • Chronic periodontitis is an inflammatory disease of the supporting tissues of teeth that is associated with specific bacteria in subgingival dental plaque. The disease has been estimated to affect around 35% of dentate adults and is a major cause of tooth loss in the Western world (1) .
  • P. gingivalis a member of the normal oral microflora of subgingival dental plague, has been implicated as one of the major opportunistic pathogens in the progression of this disease (2) .
  • P. gingivalis is a black-pigmented, asaccharolytic, Gram-negative anaerobic, cocco-bacillus, that relies on the fermentation of amino acids for energy production (3) .
  • P. gingivalis has an essential growth requirement for iron that it preferentially acquires in the form of haem, a molecule comprised of a protoporphyrin IX ring (PPIX) with a co-ordinated central ferrous atom (4) .
  • This utilization of haem as an iron source may reflect the inability of P. gingivalis to synthesize PPIX de novo (5) .
  • Haem is preferentially obtained from haemoglobin, and is acquired through the activity of the cell-surface Arg- and Lys-specific proteinase/adhesin complex (4,6,7) , possibly in conjunction with a TonB-linked outer membrane receptor, HmuR (8) .
  • P. gingivalis does not produce siderophores and lacks the ferric reductase activity usually associated with siderophore-mediated iron acquisition (9,10) .
  • P. gingivalis stores haem on its surface in the form of ⁇ -oxo bis-haem, which has inherent catalase activity that helps to protect the cell from oxidative attack (11) .
  • P. gingivalis to be able to compete with the large numbers and diversity of bacteria within the micronutrient-limiting environment of the oral cavity (12) it not only has to establish itself but also has to evade or overcome numerous host defenses.
  • compositions which would be useful in the prevention and treatment of periodontal disease it is desirable to identify agents that interfere and prevent the initial stages of the disease process.
  • the present inventors have now developed methods for identifying specific P. gingivalis proteins regulated by haem availability that can be used as suitable targets for the prevention and treatment of periodontal disease.
  • the present inventors have successfully developed methods of identifying P. gingivalis proteins regulated by haem availability that are responsible for P. gingivalis metabolism, virulence and invasion of host cells.
  • two specific internalin-like P. gingivalis proteins namely PG0350, PG1374 involved in the internalization of P. gingivalis by host cells, a hypothetical protein, PG1019 purported to be a cell surface lipoprotein and an alkyl hydroperoxide reductase protein, PG0618 have been identified as useful targets for the prevention and treatment of periodontal disease.
  • a first aspect of the invention is an isolated antigenic P. gingivalis polypeptide, the polypeptide being selected from the group consisting of:
  • a second aspect of the invention is a vaccine composition for use in raising an immune response directed against P. gingivalis in a subject, the composition comprising an effective amount of at least one polypeptide of the first aspect of the invention and a pharmaceutically acceptable carrier.
  • a third aspect of the invention is a method of preventing or treating a subject for periodontal disease comprising administering to the subject a vaccine composition according to the present invention.
  • a fourth aspect of the invention is an antibody raised against a polypeptide of the first aspect of the present invention.
  • the antibody binds specifically to the polypeptides of the present invention.
  • a fifth aspect of the invention is a composition useful in the prevention or treatment of periodontal disease, the composition comprising an antibody of the fourth aspect of the present invention and a pharmaceutically acceptable carrier.
  • a method of identifying a P. gingivalis polypeptide involved in the progression of periodontal disease comprising the steps of:
  • step a) determining the relative amount of a polypeptide or peptide thereof produced by P. gingivalis grown under haem limited conditions; and b) determining the relative amount of the polypeptide or peptide thereof produced by P. gingivalis grown under higher haem conditions than step a); wherein an increase in the amount of the polypeptide or peptide fragment thereof detected in step a) compared to step by indicates that the polypeptide is involved in the progression of periodontal disease.
  • an interfering RNA molecule comprising a double stranded region of at least 19 base pairs in each Strand wherein one of the strands of the double stranded region is complementary to a region of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO; 7 or SEQ ID NO: 8.
  • an eighth aspect of the present invention there is provided for the use of at least one polypeptide of the first aspect of the present invention in the manufacture of a medicament for the treatment of periodontal disease in a subject.
  • FIG. 1 shows a diagrammatic representation of the combined strategy used to identify proteins from P. gingivalis grown under haem-limitation.
  • the lysed cells were prefractionated into soluble and insoluble fractions using ultra-centrifugation, followed by analysis of these two fractions.
  • the separation and analysis procedure consists of two main methods; LCMS with gas phase fractionation and geLCMS.
  • FIG. 2 shows a graph indicating the codon adaptation index (CAI) distribution of the identified P. gingivalis proteome and the predicted P. gingivalis genome calculated using INCA (14) .
  • CAI codon adaptation index
  • FIG. 3 shows the analysis and identification of PG0390 based on detection of a single peptide
  • A Total ion chromatogram.
  • B Mass spectrum at 54.8 min, insert showing an enlarged ICAT peptide ion pair at 703.9 and 708.4 m/z of ratio 1:2 (L/H).
  • FIG. 4 shows the distribution of protein abundance based on ratio of haem-limitation over haem-excess (every one unit on the Log 2 scale indicates a two fold change).
  • FIG. 5 shows binding to KB cells by P. gingivalis W50 ( ⁇ ) and ECR312 ( ⁇ ).
  • Insert shows the gating of live KB cells based on forward and side scattering properties (top left), five peaks representing FITC fluorescence of hound P. gingivalis W50 to KB cells at P. gingivalis :KB cell ratios of 250 to 1250 (top right) and ECR312 (bottom right).
  • FIG. 6 shows the amino acid sequence of PG0350 protein (referred to as SEQ ID NO:1 as also indicated in the sequence listing).
  • FIG. 7 shows the amino acid sequence of PG1374 protein. (referred to as SEQ ID NO:2 as also indicated in the sequence listing).
  • FIG. 8 shows the amino acid sequence of PG1019 protein (referred to as SEQ ID NO:3 as also indicated in the sequence listing).
  • FIG. 9 shows the amino acid sequence of PG0618 protein (referred to as SEQ ID NO:4 as also indicated in the sequence listing).
  • the present invention advantageously provides the identification of P. gingivalis proteins regulated by haem availability as useful targets for the prevention and treatment of periodontal disease.
  • the invention provides the identification of P. gingivalis proteins upregulated by haem-limitation as useful targets for the prevention and treatment of periodontal disease.
  • the invention provides an isolated antigenic P. gingivalis polypeptide, the polypeptide being selected from the group consisting of:
  • the isolated antigenic polypeptide is 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4.
  • the isolated antigenic polypeptide comprises an amino acid sequence comprising at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids identical to a contiguous amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4.
  • the antigenic polypeptides comprise amino acid sequences that compose the hydrophilic, surface-exposed regions of the PG1374 or PG0350 or PG1019 or PG0618 protein.
  • polypeptides, proteins, and polypeptides are used interchangeably herein.
  • the polypeptides of the present invention can include recombinant polypeptides such as fusion polypeptides. Methods for the production of a fusion polypeptide are well-known to those skilled in the art.
  • altered polypeptides can be either naturally occurring (that is to say, purified or isolated from a natural source) or synthetic (for example, by site-directed metagenesis on the encoding DNA). It is intended that such altered polypeptides which have at least 85%, preferably at least 90%, 95%, 96%, 97%, 98% or 99% identity with the sequences set out in the Sequence Listing are within the scope of the present invention. Antibodies raised against these altered polypeptides will also bind to the polypeptides having one of the sequences set out in the Sequence Listings.
  • isolated polypeptide refers to a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs or the polypeptide or peptide may be synthetically synthesised.
  • the polypeptide is also separated from substances, for example, antibodies or gel matrix, for example, polyacrylamide, which are used to purify it.
  • the polypeptide constitutes at least 10%, 20%, 50%, 70%, and 80% of dry weight of the purified preparation.
  • the preparation contains a sufficient amount of polypeptide to allow for protein sequencing (i.e. at least 1, 10, or 100 mg).
  • the isolated polypeptides described herein may be purified by standard techniques, such as column chromatography (using various matrices which interact with the protein products, such as ion exchange matrices, hydrophobic matrices and the like), affinity chromatography utilizing antibodies specific for the protein or other ligands which bind to the protein.
  • an “antigenic polypeptide” used herein is a moiety, such as a polypeptide, analog or fragment thereof, that is capable of binding to a specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.
  • the antigenic polypeptide comprises an immunogenic component that is capable of eliciting a humoral and/or cellular immune response in a host animal.
  • a “contiguous amino acid sequence” as used herein refers to a continuous stretch of amino acids.
  • amino acid sequence identities or similarities may be calculated using the GAP programme and/or aligned using the PILEUP programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wis., United States of America (20) .
  • the GAP programme utilizes the algorithm of Needleman and Wunsch (21) to maximise the number of identical/similar residues and to minimise the number and length of sequence gaps in the alignment.
  • the Clustal W programme (22) is used.
  • the present invention also provides a vaccine composition for use in raising an immune response directed against P. gingivalis in a subject, the composition comprising an effective amount of at least one polypeptide of the first aspect of the invention and a pharmaceutically acceptable carrier.
  • the vaccine composition of the present invention preferably comprises an antigenic polypeptide that comprises at least one antigen that can be used to confer immunity against P. gingivalis .
  • the subject treated by the method of the invention may be selected from, but is not limited to, the group consisting of humans, sheep, cattle, horses, bovine, pigs, poultry, dogs and cats.
  • the subject is a human.
  • An immune response directed against P. gingivalis is achieved in a subject, when the subject's immune system produces antibodies against the specific antigenic polypeptides.
  • the vaccine composition is preferably administered to a subject to induce immunity to P. gingivalis and thereby prevent periodontal disease.
  • the term “effective amount” as used herein means a dose sufficient to elicit an immune response against P. gingivalis . This will vary depending on the subject and the level of P. gingivalis infection and ultimately will be decided by the attending scientist, physician or veterinarian.
  • the vaccine composition of the present invention comprises a suitable pharmaceutically-acceptable carrier, such as diluent and/or adjuvant suitable for administration to a human or animal subject.
  • the vaccine preferably comprises a suitable adjuvant for delivery orally by nasal spray, or by injection to produce a specific immune response against P. gingivalis .
  • a vaccine of the present invention can also be based upon a recombinant nucleic acid sequence encoding an antigenic polypeptide of the present invention, wherein the nucleic acid sequence is incorporated into an appropriate vector and expressed in a suitable transformed host (e.g. E. coli, Bacillus subtilis, Saccharomyces cerevisiae , COS cells, CHO cells and HeLa cells) containing the vector.
  • a suitable transformed host e.g. E. coli, Bacillus subtilis, Saccharomyces cerevisiae , COS cells, CHO cells and HeLa cells
  • the vaccine can be produced using recombinant DNA methods as illustrated herein, or can be synthesized chemically from the amino acid sequence described in the present invention. Additionally, according to the present invention, the antigenic polypeptides may be used to generate P. gingivalis antisera useful for passive immunization against periodontal disease and infections caused by P. gingivalis.
  • adjuvants known those skilled in the art are commonly 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 Freunds adjuvant (IFA), Adjuvant 65 (containing peanut oil, mannide monooleate and aluminium monostrearate), oil emulsions, Ribi adjuvant, the pluronic polyols, polyamines, Avridine, Quil A, saponin, MPL, QS-21, and mineral gels such as aluminium salts.
  • the vaccine may include conventional pharmaceutically acceptable carriers, excipients, fillers, buffers or diluents as appropriate.
  • One or more doses of the vaccine containing adjuvant may be administered prophylactically to prevent periodontal disease or therapeutically to treat already present periodontal disease.
  • the preparation is combined with a mucosal adjuvant and administered via the oral or nasal mute.
  • mucosal adjuvants are cholera toxin and heat labile E. coli toxin, the non-toxic B sub-units of these toxins, genetic mutants of these toxins which have reduced toxicity.
  • Other methods which may be utilised to deliver the antigenic polypeptides orally or nasally include incorporation of the polypeptides into particles of biodegradable polymers (such as acrylates or polyesters) by micro-encapsulation to aid uptake of the microspheres from the gastrointestinal tract or nasal cavity and to protect degradation of the proteins.
  • Liposomes, ISCOMs, hydrogels are examples of other potential methods which may be further enhanced by the incorporation of targeting molecules such as LTB, CTB or lectins (mannan, chitin, and chitosan) for delivery of the antigenic polypeptides to the mucosal immune system.
  • targeting molecules such as LTB, CTB or lectins (mannan, chitin, and chitosan)
  • the vaccine may include conventional pharmaceutically acceptable carriers, excipients, fillers, coatings, dispersion media, antibacterial and antifungal agents, buffers or diluents as appropriate.
  • Another mode of this embodiment provides for either, a live recombinant viral vaccine, recombinant bacterial vaccine, recombinant attenuated bacterial vaccine, or an inactivated recombinant viral vaccine which is used to protect against infections caused by P. gingivalis .
  • Vaccinia virus is the best known example, in the art, of an infectious virus that is engineered to express vaccine antigens derived from other organisms.
  • the recombinant live vaccinia virus which is attenuated or otherwise treated so that it does not caused disease by itself, is used to immunise the host. Subsequent replication of the recombinant virus within the host provides a continual stimulation of the immune system with the vaccine antigens such as the antigenic polypeptides, thereby providing long lasting immunity.
  • live vaccine vectors include: adenovirus, cytomegalovirus, and preferably the poxviruses such as vaccinia (24) and attenuated salmonella strains (25-28) .
  • Live vaccines are particularly advantageous because they continually stimulate the immune system which can confer substantially long-lasting immunity.
  • the live vaccine itself may be used in a protective vaccine against P. gingivalis .
  • the live vaccine can be based on a bacterium that is a commensal inhabitant of the oral cavity. This bacterium can be transformed with a vector carrying a recombinant inactivated polypeptide and then used to colonise the oral cavity, in particular the oral mucosa.
  • the expression of the recombinant protein will stimulate the mucosal associated lymphoid tissue to produce neutralising antibodies.
  • the genes encoding the polypeptides may be inserted into the vaccinia virus genomic DNA at a site which allows for expression of epitopes but does not negatively affect the growth or replication of the vaccinia virus vector.
  • the resultant recombinant virus can be used as the immunogen in a vaccine formulation.
  • the same methods can be used to construct an inactivated recombinant viral vaccine formulation except the recombinant virus is inactivated, such as by chemical means known in the art, prior to use as an immunogen and without substantially affecting the immunogenicity of the expressed immunogen.
  • immunisation may be passive, i.e. immunisation comprising administration of purified immunoglobulin containing an antibody against a polypeptide of the present invention.
  • the antigenic polypeptides used in the methods and compositions of the present invention may be combined with suitable excipients, such as emulsifiers, surfactants, stabilisers, dyes, penetration enhancers, anti-oxidants, water, salt solutions, alcohols, polyethylene glycols, gelatine, lactose, magnesium sterate and silicic acid.
  • suitable excipients such as emulsifiers, surfactants, stabilisers, dyes, penetration enhancers, anti-oxidants, water, salt solutions, alcohols, polyethylene glycols, gelatine, lactose, magnesium sterate and silicic acid.
  • the antigenic polypeptides are preferably formulated as a sterile aqueous solution.
  • the vaccine compositions of the present invention may be used to complement existing treatments for periodontal disease.
  • a third aspect of the invention is a method of preventing or treating a subject for periodontal disease comprising administering to the subject a vaccine composition according to the present invention.
  • Periodontal diseases range from simple gum inflammation to serious disease that results in major damage to the soft tissue and bone that support the teeth.
  • Periodontal disease includes gingivitis and periodontitis.
  • Bacteria, mainly Gram-negative species including P. gingivalis cause inflammation of the gums that is called “gingivitis.” In gingivitis, the gums become red, swollen and can bleed easily. When gingivitis is not treated, it can advance to “periodontitis” (which means “inflammation around the tooth.”).
  • gingivitis which means “inflammation around the tooth.”.
  • gingivitis which means “inflammation around the tooth.”.
  • gingivitis which means “inflammation around the tooth.”.
  • gingivitis which means “inflammation around the tooth.”.
  • gingivitis which means “inflammation around the tooth.”.
  • gingivitis which means “inflammation around the tooth.”.
  • gingivitis which means “inflammation around the tooth.”.
  • a fourth aspect of the invention is an antibody raised against a polypeptide of the first aspect of the present invention.
  • the antibody is specifically directed against the polypeptides of the present invention.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, diabodies, triabodies and antibody fragments.
  • the antibodies of the present invention are preferably able to specifically bind to an antigenic polypeptide as hereinbefore described without cross-reacting with antigens of other polypeptides.
  • binding specifically to is intended to refer to the binding of an antigen by an immunoglobulin variable region of an antibody with a dissociation constant (Kd) of 1 ⁇ M or lower as measured by surface plasmon resonance analysis using, for example a BIAcoreTM surface plasmon resonance system and BIAcoreTM kinetic evaluation software (eg. version 2.1).
  • Kd dissociation constant
  • the affinity or dissociation constant (Kd) for a specific binding interaction is preferably about 500 nM to about 50 pM, more preferably about 500 nM or lower, more preferably about 300 nM or lower and preferably at least about 300 nM to about 50 pM, about 200 nM to about 50 pM, and more preferably at least about 100 nM to about 50 pM, about 75 nM to about 50 pM, about 10 nM to about 50 pM.
  • binding fragments of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment which consists of a VH domain, or a VL domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • a F(ab′)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • a Fd fragment consisting of the VH and CH1 domain
  • the two domains of the Fv fragment, VL and VH are coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL, and VH regions pair to form monovalent molecules (known as single chain Fv (scFv).
  • scFv single chain Fv
  • Other forms of single chain antibodies, such as diabodies or triabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.
  • Antibodies and antibody fragments may be produced in large amounts by standard techniques (eg in either tissue culture or serum free using a fermenter) and purified using affinity columns such as protein A (e.g. for murine Mabs), Protein G (eg for rat Mabs) or MEP HYPERCEL (eg for IgM and IgG Mabs).
  • protein A e.g. for murine Mabs
  • Protein G e.g for rat Mabs
  • MEP HYPERCEL eg for IgM and IgG Mabs.
  • Humanized antibodies may be prepared according to procedures in the literature (29,30) , The recently described “gene conversion metagenesis” strategy for the production of humanized monoclonal antibody may also be employed in the production of humanized antibodies (31) .
  • techniques for generating the recombinant phase library of random combinations of heavy and light regions may be used to prepare recombinant antibodies (32) .
  • the present invention also provides a composition useful in the prevention or treatment of periodontal disease, the composition comprising an antagonist of a P. gingivalis polypeptide of the first aspect of the present invention and a pharmaceutically acceptable carrier, wherein the antagonist inhibits P. gingivalis infection.
  • the term “antagonist” refers to a nucleic acid, peptide, antibody, ligands or other chemical entity which inhibits the biological activity of the polypeptide of interest.
  • a person skilled in the art would be familiar with techniques of testing and selecting suitable antagonists of a specific protein, such techniques would including binding assays.
  • Possible antagonists of PG0350, PG1374, PG1019 and PG0618 are preferably antibodies, either monoclonal or polyclonal, which will inhibit the binding of these proteins to host cells or other substrates or they may be proteins or peptides that interfere with the binding of these proteins.
  • the antibodies and antagonists of the present invention have a number of applications, for example, they can be used as antimicrobial preservatives, in oral care products (toothpastes and mouth rinses) for the control of dental plaque and suppression of pathogens associated with dental caries and periodontal diseases.
  • the antibodies and antagonists of the present invention may also be used in pharmaceutical preparations (eg, topical and systemic anti infective medicines).
  • a method of identifying a P. gingivalis polypeptide involved in the progression of periodontal disease comprising the steps of:
  • step a) determining the relative amount of a polypeptide or peptide thereof produced by P. gingivalis grown under haem limited conditions; and b) determining the relative amount of the polypeptide or peptide thereof produced by P. gingivalis grown under higher haem conditions than step a); wherein an increase in the amount of the polypeptide or peptide fragment thereof detected in step a) compared to step b) indicates that the polypeptide is involved in the progression of periodontal disease.
  • the concentration of haemin is about 0.1 ⁇ g/ml to about 0.5 ⁇ g/ml.
  • Haem limiting conditions are achieved when the cell density of the P. gingivalis cells is significantly lower than that observed under the growth conditions of step (h) of the method of the invention.
  • the higher haem conditions of step (b) is preferably achieved using a concentration of haemin of above 5 mg/ml.
  • a comparison of the relative amounts of a polypeptide in the haem limited and higher haem conditions can be preferably determined by using a differential proteomic approach.
  • the amount of a polypeptide is determined by qualitative proteomic analysis commonly used in the art, such as but not limited to, a combined strategy of in-solution and in-gel digestion and LC-MS/MS, analysis using stable isotope labelling strategies (ICAT) in combination with MS.
  • ICAT stable isotope labelling strategies
  • the isolated antigenic P. gingivalis polypeptides identified according to the method defined in the sixth aspect of the invention can be used as targets for treating and preventing periodontal disease.
  • the isolated polypeptides can be used to develop vaccine compositions against P. gingivalis , for instance P. gingivalis infection, such as periodontal disease.
  • the present invention also provides interfering RNA molecules which are targeted against the mRNA molecules encoding the polypeptides of the first aspect of the present invention. Accordingly, in a seventh aspect of the present invention there is provided an interfering RNA molecule, the molecule comprising a double stranded region of at least 19 base pairs in each strand wherein one of the strands of the double stranded region is complementary to a region of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8.
  • RNA interference So called RNA interference or RNAi is well known and further information regarding RNAi is provided in Hannon (2002) Nature 418: 244-251, and McManus & Sharp (2002) Nature Reviews; Genetics 3(10): 737-747, the disclosures of which are incorporated herein by reference.
  • the present invention also contemplates chemical modification(s) of siRNAs that enhance siRNA stability and support their use in vivo (see for example, Shen et al. (2006) Gene Therapy 13: 225-234). These modifications might include inverted abasic moieties at the 5′ and 3′ end of the sense strand oligonucleotide, and a single phosphorthioate linkage between the last two nucleotides at the 3′ end of the antisense strand.
  • the double stranded region of the interfering RNA comprises at least 20, preferably at least 25, and most preferably at least 30 base pairs in each strand of the double stranded region.
  • the present invention also provides a method of treating a subject for periodontal disease comprising administering to the subject at least one of the interfering RNA Molecules of the invention.
  • P. gingivalis W50 (ATCC 53978) was obtained from the culture collection of the Centre for Oral Health Science, The University of Melbourne. Chemicals used were ultra high purity except for MS work where LC MS grade reagents were used (Sigma, Reidel-de Ha ⁇ umlaut over (c) ⁇ n).
  • P. gingivalis W50 was grown in continuous culture using a Bioflo 110 fermenter/bioreactor (New Brunswick Scientific) with a 400 mL working volume.
  • the growth medium was 37 g/mL brain heart infusion medium (Oxoid) supplemented with 5 mg/mL filler sterilized cysteine hydrochloride, 5.0 ⁇ g/mL haemin (haem-excess) or 0.1 ⁇ g/mL haemin (haem-limited). Growth was initiated by inoculating the culture vessel with a 24 h batch culture (100 mL) of P. gingivalis grown in the same medium (haem-excess).
  • the medium reservoir pump was turned on and the medium flow adjusted to give a dilution rate of 0.1 h ⁇ 1 (mean generation time (MGT) of 6.9 h).
  • MCT mean generation time
  • the temperature of the vessel was maintained at 37° C. and the pH at 7.4 ⁇ 0.1.
  • the culture was continuously gassed with 5% CO 2 in 95% N 2 .
  • Cells were harvested during steady state growth, washed three times with wash buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl 2 ) at 5000 g for 30 min and disrupted with 3 passes through a French Pressure Cell (SLM, AMINCO) at 138 MPa.
  • SLM, AMINCO French Pressure Cell
  • the lysed cells were then centrifuged at 2000 g for 30 min to remove unbroken cells followed by ultracentrifugation at 100000 g, producing a soluble (supernatant) and membrane fraction. All fractions were carried out on
  • Non-quantitative proteome analysis was carried out using two methods, in-solution digestion with trypsin followed by LCMS with gas phase fractionation (GPF) and in-gel digestion followed by LCMS (geLC-MS) as part of a combined strategy ( FIG. 1 ).
  • in-solution digestion method protein was boiled at 95° C. for 3 min, precipitated with TCA (16%) and resuspended in solubilization buffer (8 M Urea, 50 mM Tris-HCl pH 8.3, 5 mM EDTA, 0.05% SDS). Protein concentration was determined with a BCA, protein reagent (Pierce) and adjusted to 2 ⁇ g/ ⁇ L.
  • Reduction was carried out with 1 in M DTT for 30 min and alkylation using 10 mM iodoacetamide for 60 min.
  • the solution was diluted with water to a final concentration of 1 M urea before digestion.
  • Digestion was carried out using sequencing-grade modified trypsin (Promega) at a ratio of 1:100 w/w trypsin to protein at 37° C. for 16 h. The digestion was terminated by formic acid addition to a final concentration of 1% v/v.
  • Peptides were then desalted using Sep-Pak C18 cartridges (Waters), dried using a vacuum centrifuge (Thermosavant) and resuspended in 5% acetonitrile in 0.1% TFA. An amount of peptide equivalent to 2 ⁇ g was injected for each LC-MS/MS analysis.
  • the reduction solution was then replaced with 55 mM of iodoacetamide in 25 mM ABC and incubated for 45 min.
  • the gel cubes were washed twice in 50 mM ABC and dehydrated with 100% ethanol.
  • Thirty ⁇ L of modified sequencing-grade trypsin at a concentration of 5 ⁇ g/mL in 25 mM ABC buffer and 1 mM CaCl 2 was added and incubated at 4° C. for 30 min. Excess trypsin solution was removed and 15 ⁇ L of 25 mM ABC buffer was added. Digestion was carried out overnight at 37° C. and terminated by TFA addition to a final concentration of 0.1% v/v. The supernatant was then transferred to an eppendorf tube.
  • Protein labelling and separation were based on the geLC-MS/MS approach (33) using the cleavable ICAT reagent (Applied Biosystems). Protein was first precipitated using TCA (16%) and solubilised with 6 M urea, 5 mM EDTA, 0.05% SDS and 50 mM Tris-HCl pH 8.3. Protein concentration was determined using the BCA protein reagent and adjusted to 1 mg/ml. 100 ⁇ g of protein from each growth condition was individually reduced using 2 ⁇ L of 50 mM Tris(2-carboxy-ethyl)phosphine hydrochloride for 1 h at 37° C.
  • Reduced protein from the haem-limitation growth condition was then alkylated with the ICAT heavy reagent and protein from haem-excess growth condition with the ICAT light reagent.
  • the two samples were then combined and subjected to SDS-PAGE on a precast Novex 10% NUPAGE gel (Invitrogen).
  • the gel was stained for 5 min using SimplyBlueTM SafeStain (Invitrogen) followed by destaining with water.
  • the gel lane was then excised into 20 sections from the top of the gel to the dye front.
  • the excised sections were further diced into 1 mm 3 cubes and in-gel digested overnight and extracted twice according to the above procedure.
  • the pooled supernatant was dried under reduced vacuum to about 50 ⁇ L followed by mixing with 500 ⁇ L, of affinity load buffer before loading onto the affinity column as per manufacturer's instruction (Applied Biosystems).
  • Eluted peptides were dried and the biotin tag cleaved with neat TFA at 37° C. for 2 h. followed by drying under reduced vacuum.
  • the dried samples were suspended in 35 ⁇ L of 5% acetonitrile in 0.1% TFA.
  • MS was carried Out using an Esquire HCT ion trap mass spectrometer (Bruker Daltonics) coupled to an UltiMate Nano LC system (LC Packings—Dionex). Separation was achieved using a LC Packings reversed phase column (C18 PepMap100, 75 ⁇ m i.d. ⁇ 15 cm, 3 ⁇ m, 100 ⁇ ), and chilled in 0.1% formic acid with the following acetonitrile gradient: 0-5 min (0%), 5-10 min (0-10%), 10-100 min (10-50%), 100-120 min (50-80%), 120-130 min (80-100%).
  • the LC output was directly interfaced to the nanospray ion source.
  • MS acquisitions were performed under an ion charge control of 100000 in the m/z range of 300-1500 with maximum accumulation time of 100 ms.
  • GPF three additional m/z ranges (300-800, 700-1200 and 1100-1500) were used to select for precursor ions and each m/z range was carried out in duplicate to increase the number of peptides identified.
  • MS/MS acquisition was obtained over a mass range from 100-3000 m/z and was performed on up to 10 precursors for initial complete proteome analysis and 3 for ICAT analysis for the most intense multiply charged ions with an active exclusion time of 2 rain.
  • Peak lists were generated using DataAnalysis 3.2 (Bruker Daltonics) using the Apex peak finder algorithm with a compound detection threshold of 10000 and signal to noise threshold of 5. A global charge limitation of +2 and +3 were set for exported data. Protein identification was achieved using the MASCOT search engine (MASCOT 2.1.02, Matrix Science) on MS/MS data queried against the P. gingivalis database obtained from The Institute for Genomic Research (TIGR) website (www.tigr.org).
  • peptides with a probability based Mowse score corresponding to a p-value of at most 0.05 were regarded as positively identified, where the score is ⁇ log ⁇ 10(log(P)) and P is the probability that the observed match is a random event ii) where only one peptide was used in the identification of a specific protein and the MASCOT score was below 30, manual verification of the spectra was performed.
  • the ratio of isotopically heavy 13 C to light 12 C ICAT labelled peptides was determined using a script from DataAnalysis (Bruker Daltonics) and verified manually based on measurement of the monoisotopic peak intensity (signal intensity and peak area) in a single MS spectrum.
  • the minimum ion count of parent ions used for quantification was 2000 although >96% of both heavy and light precursor ions were >10000.
  • gingivalis from genebank (ftp ://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/Porphyromonas_gingivalis_W83/) using the program INteractive Codon Analysis 1.12a (http://www.bioinfo-hr.org/inca, (14) with ribosomal proteins and tRNA synthases being defined as highly expressed genes. Operon prediction was carried out from the Microbesonline website (http://microbesonline.org (36) .
  • P. gingivalis W50 Open Reading Frame PG1374 potentially encodes an immunoreactive 47 KDa antigen (PG97) based on the P. gingivalis W83 genome (www.tigr.org).
  • PG97 immunoreactive 47 KDa antigen
  • PG1374 mutant a 672 bp upstream fragment of the PG1374 gene with flanking ApaI and AatII restriction sites (underlined) was generated from the W50 genomic DNA by PCR with primers ECR312ApaI-For (5′-AGA GGGCCC TAGCAATCATTGCATTGCT-3′) and ECR312AatII-Rev (5′-TGC GACGTC GTGTTACCAATAGAGGATT-3′).
  • This fragment was cloned into AatII and BamHI sites on pAL30, pGem®T-easy (Promega) containing a subcloned ermF cassette (37) to create pAL36.
  • a 565 bp fragment downstream of PG1374 with flanking PstI and NdeI restrictions sites was amplified with ECR312PstI-For2 (5′-TGA CTGCAG GCTTTCGACCTTGGATCTT-3′) and ECR312NdeI-Rev2 (5′-TCG CATATG AAGAAATAAGTGCCGTCGG-3′) primers, and cloned into PstI and NdeI restrictions sites in pAL36.
  • the resulting plasmid having ermF cassette flanked with upstream and downstream fragments of the PG1374 open reading frame (designated as pAL36.1) was linearized with Scar and transformed into P. gingivalis W50 as previously described (38) .
  • Transformed cells were selected after 7 days of incubation at 37° C. under anaerobic conditions on HBA plate containing 10 ⁇ g mL ⁇ 1 erythromycin. Confirmation of DNA integration was performed by PCR analysis and the resulting mutant was designated as ECR312.
  • an antibiotic protection assay was carried out as described previously in a 24-well cell culture plate (39) . Briefly, a fixed number of P. gingivalis cells were allowed to invade a KB monolayer ( ⁇ 10 5 cells in each well). Non invaded or adhered cells were killed by further incubation for 1 h with gentamicin (300 ⁇ g/mL) and metronidazole (200 ⁇ g/mL). Colony forming units of invaded bacteria were then enumerated on horse blood agar plates.
  • the cell binding assay was carried out as described previously (40) . Briefly, P. gingivalis was first grown to mid log phase to a cell density of ⁇ 2.9 ⁇ 10 9 cells/mL. The cells were then washed followed by labelling with 500 ⁇ g of fluorescein isothiocyanate (FITC) (Invitrogen) resuspended in 500 ⁇ L DMSO followed by incubation at 37° C. for 45 min with shaking. After incubation, the cells were further washed, resuspended in incomplete Earl's minimum essential medium (JRH Biosciences) and the P.
  • FITC fluorescein isothiocyanate
  • gingivalis cells adjusted based on cell counts using a FACSCaliber flow cytometer (Becton Dickinson, San Jose, Calif.). The green emission of FITC was measured with a 525-nm filter (FL1). The multiparametric data were analyzed using CellQuest software (Becton Dickinson, San Jose, Calif.). All measurements were done in duplicate, and for quantitation of FITC fluorescence, mean fluorescence intensity (MFI) values were used.
  • FACSCaliber flow cytometer Becton Dickinson, San Jose, Calif.
  • Binding of the wild type P. gingivalis and ECR312 was carried out in parallel by inoculating 200 ⁇ L of cell suspension onto the KB cells at 5% CO 2 atmosphere at 37° C. for 40 min. Following incubation the supernatant containing the KB cells and bacteria were transferred to a 1.5 mL tube. The remaining bound cells were then detached off the well with 200 ⁇ L of Trypsin-EDTA mixture (JRH Bioscience) for 5 min at 37° C. and pooled with the corresponding collected supernatants. 500 ⁇ L of complete EMEM was then added to inactivate the trypsin followed by three washes and final suspension in 1 mL PBS. The bound cells were counted on the flow cytometer as described earlier.
  • proteome of P. gingivalis grown under haem-limitation was extensively analysed by two different approaches. Using in-solution digestion followed by LCMS with gas phase fractionation, 344 proteins were identified. In the geLCMS approach, 385 proteins were identified while 247 proteins were found by both approaches. With the combined strategy a total of 478 proteins were identified (see Table 1) with an estimated false positive rate of 0.4% calculated from searches against the P. gingivalis reverse database. 77.0% of all proteins were identified by ⁇ 2 unique peptides or by ⁇ 2 identical peptides from independent LCMS runs (from different m/z ranges or SDS PAGE bands).
  • the 478 identified proteins represent ⁇ 25% of all the 1988 assigned protein-encoding genes identified by whole-genome analysis (43) . Although a quarter of the total predicted proteome was identified this figure is higher if the actual number of genes expressed during any one growth condition is taken into account. In Pseudomonas aeruginosa and Bacillus subtilis the percentage of total ORFs transcribed during a single growth condition was estimated to be 60% and 40%, respectively (44,45) . By examining the duty cycle limitation of their mass spectrometers Zhang and co-workers (46) estimated that around 60% of P. gingivalis predicted ORFs were expressed under their growth condition. Therefore based on these figures between 41-62% of P. gingivalis proteins expressed under the present growth conditions have been identified. The functional classification of the 478 identified proteins is shown in Table 1.
  • TMHMM Transmembrane Hidden Markov Model
  • CAD Codon Adaptation Index
  • the functional classes of proteins with the highest percentage of identified proteins are those involved in energy metabolism (Table 1), typically those involved in fermentation (95%, CAI 0.70-0.80), glycolysis (82%, CAI 0.71-0.83) and metabolism of amino acids and amines (81%, CAI 0.71-0.84). This was largely expected as essential proteins involved in basic metabolic functions such as energy metabolism have been shown to be very abundant in the bacterial cell. Most importantly almost 90% of these proteins are predicted to be in the cytoplasm, which also made detection easier compared with membrane proteins.
  • the functional classes of proteins represented least are those involved in transpositioning, hypothetical proteins and regulatory functions.
  • P. gingivalis is known to be asaccharolytic (3) although the genome contains putative ORFs for all enzymes of the glycolytic pathway (43) .
  • the poor utilization of this pathway has been attributed to the glucose kinase gene being interrupted by an insertion element (43) .
  • glucose kinase or another glycolysis-specific enzyme, phosphofructokinase was not identified.
  • all enzymes involved in gluconeogenesis were found, suggesting glucose necessary for processes such as polysaccharide biosynthesis may be derived via this pathway.
  • the geLCMS approach was chosen, as the in-solution ICAT method was unsatisfactory due to the presence of strong interfering triply charged ions. The presence of these triply charged ions resulted in very low number of protein identifications.
  • the ICAT labelled soluble and insoluble protein fractions were therefore independently separated by SDS-PACE and each gel lane divided into 20 sections for in-get tryptic digestion followed by affinity purification and LCMS. In total 142 proteins were identified. No matches to the reverse database were obtained indicating a low level of false positive identification.
  • PG1374 and PG0350 belong to a new class of cysteine containing protein with leucine rich repeat domains similar to the L. monocytogenes internalin protein. In1J (52) .
  • L. monocytogenes there are at least fifteen members of the internalin family and all have been found to share certain structural features consisting of a signal peptide, N-terminal leucine rich repeat domain followed by a conserved inter-repeat region. Many of these proteins are involved in the cellular invasion process (53) . It has not been demonstrated why multiple internalins exist, but they are proposed to confer tropism toward different cell types (54) .
  • PG1374 and PG0350 both possesses a signal peptide and are part of the novel class of up to 34 cell surface-located outer membrane proteins that have no significant sequence similarities apart from a conserved C-Terminal Domain (CTD) of approximately 80 residues (56) .
  • CCD conserved C-Terminal Domain
  • PG1374 is strongly immunogenic when probed with sera from human periodontitis patients (57) which further suggests it to be involved in cell surface protein interactions.
  • PG1019 was observed to be 25 times more abundant when the bacterium was grown under haem-limitation. Bioinformatic analyses suggest that PG1019 is a lipoprotein that is encoded by a gene located immediately upstream of a gene encoding a putative outer membrane receptor protein (PG1020) in a predicted operon. Multiple alignment (not shown) of PG1020 with known P.
  • TonB-linked outer membrane receptors shows the presence of a putative TonB box (residues 118-126), that is one of the characteristics of TonB-linked receptors (67) and a conserved region (residues 236-272) which Simpson and co-workers (68) refer to as the TonB box IV region.
  • TonB-linked outer membrane receptors have been implicated with many iron, iron complex and other micronutrient uptake systems. The high abundance of PG1019 under haem limited growth conditions would be consistent with this protein being an accessory lipoprotein to a Ton-B linked system involved in the transport of iron/iron complexes into the cell or the sensing of environmental iron or iron complexes, although this remains to be demonstrated.
  • P. gingivalis FB1 a transcriptomic analysis using custom made P. gingivalis DNA microarrays of P. gingivalis W50 compared to a mutant lacking a function feoB1 gene ( P. gingivalis FB1) was performed.
  • the wild type W50 and FB1 mutant were both grown in continuous culture in haem excess conditions.
  • P. gingivalis FB1 has approximately half the cellular iron content of the wild type W50 (69) . Genes that show an increase in transcription are therefore likely to be upregulated in response to the decrease in intracellular or environmental iron content.
  • Both PG1019 and PG1020 were significantly upregulated to similar levels in the FB1 mutant compared to the wild type.
  • PG1019 showed a Log 2 increase of 2.46 and PG1020 showed a Log 2 increase of 2.33 at a significance level of P ⁇ 0.05 in biological replicates, this is further evidence that these genes are located in an operon. Further a separate transcriptomic DNA microarray analysis of P. gingivalis W50 indicated that there was little or no expression of the PG1019 gene during haem-excess growth.
  • OxyR an oxygen sensitive transcriptional activator also plays a role in the expression of alkyl hydroperoxide during anaerobic growth (71) where a P. gingivalis OxyR ⁇ mutant shows decrease of 16 fold in gene expression. More recently Duran-Pinedo and co-workers also demonstrated the positive regulation of aphC expression by the RprY response regulator.

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