Abstract:
The present invention relates to isolated  Porphyromonas gingivalis  polypeptides and nucleotides. The polypeptides include an amino acid sequence selected from the group consisting of: SEQ. ID. NO. 110; SEQ. ID. NO. 111; SEQ. ID. NO. 112; SEQ. ID. NO. 113; SEQ ID NO: 120; SEQ. ID. NO. 123; SEQ. ID. NO. 124; SEQ. ID. NO. 125; SEQ. ID. NO. 130; SEQ. ID. NO. 131; SEQ. ID. NO. 132; SEQ. ID. NO. 133; SEQ. ID. NO.135; SEQ. ID. NO. 136; SEQ. ID. NO. 137; SEQ. ID. NO. 138; SEQ. ID. NO. 143; SEQ. ID. NO. 144; SEQ. ID. NO. 145; SEQ. ID. NO. 146; SEQ. ID. NO. 147; SEQ. ID. NO. 148; and amino acid sequences at least 95% identical thereto.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation-in-part of application Ser. No. 11/589,261, filed Oct. 30, 2006, which is a continuation of application Ser. No. 09/581,286, filed Jun. 28, 2000, which is a 317 of PCT/AU98/01023 filed Dec. 10, 1998, the entire contents of each of these applications is incorporated herein by reference. In particular, the entire contents of application Ser. No. 11/589,261, pending, is incorporated herein by reference, including specifically the sequence ID numbers: SEQ. ID. NO. 1 to SEQ ID NO: 721. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to  Porphyromonas gingivalis  nucleotide sequences,  P. gingivalis  polypeptides and probes for detection of  P. gingivalis.  The  P. gingivalis  polypeptides and nucleotides can be used in compositions for use in raising an immune response in a subject against  P. gingivalis  and treating or preventing or reducing the severity of the condition known as periodontitis. 
       BACKGROUND OF THE INVENTION 
       [0003]    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&#39;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  P. 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.  These findings in both animals and humans suggest a major role for  P. gingivalis  in the development of adult periodontitis. 
         [0004]      P. gingivalis  is a black-pigmented, anaerobic, asaccharolytic, 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 haeme or its Fe(III) oxidation product haemin and when grown under conditions of excess haemin 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. 
         [0005]    In order to develop an efficacious and safe vaccine to prevent, eliminate or reduce  P. gingivalis  colonisation it is necessary to identify and produce antigens that are involved in virulence that have utility as immunogens possibly through the generation of specific antibodies. Whilst it is possible to attempt to isolate antigens directly from cultures of  P. gingivalis  this is often difficult. For example as mentioned above,  P. gingivalis  is a strict anaerobe and can be difficult to isolate and grow. It is also known that, for a number of organisms, when cultured in vitro that many virulence genes are down regulated and the encoded proteins are no longer expressed. If conventional chemistry techniques were applied to purify vaccine candidates potentially important (protective) molecules may not be identified. With DNA sequencing, as the gene is present (but not transcribed) even when the organism is grown in vitro it can be identified, cloned and produced as a recombinant DNA protein. Similarly, a protective antigen or therapeutic target may be transiently expressed by the organism in vitro or produced in low levels making the identification of these molecules extremely difficult by conventional methods. 
         [0006]    With serological identification of therapeutic targets one is limited to those responses which are detectable using standard methods such as Western Blotting or ELISA. The limitation here is the both the level of response that is generated by the animal or human and determining whether this response is protective, damaging or irrelevant. No such limitation is present with a sequencing approach to the identification of potential therapeutic or prophylactic targets. 
         [0007]    It is also well known that  P. gingivalis  produces a range of broadly active proteases (University of Melbourne International Patent Application No PCT/AU 96/00673, U.S. Pat. Nos. 5,475,097 and 5,523,390), which make the identification of intact proteins difficult because of their degradation by these proteases. 
       SUMMARY OF THE INVENTION 
       [0008]    The present inventors have attempted to isolate  P. gingivalis  nucleotide sequences which can be used for recombinant production of  P. gingivalis  polypeptides and to develop nucleotide probes specific for  P. gingivalis.  The DNA sequences listed below have been selected from a large number of  P. gingivalis  sequences according to their indicative potential as vaccine candidates. This intuitive step involved comparison of the deduced protein sequence from the  P. gingivalis  DNA sequences to the known protein sequence databases. Some of the characteristics used to select useful vaccine candidates include; the expected cellular location, such as outer membrane proteins or secreted proteins, particular functional activities of similar proteins such as those with an enzymatic or proteolytic activity, proteins involved in essential metabolic pathways that when inactivated or blocked may be deleterious or lethal to the organism, proteins that might be expected to play a role in the pathogenesis of the organism eg. red cell lysis, cell agglutination or cell receptors and proteins which are paralogues to proteins with proven vaccine efficacy. 
         [0009]    In a first aspect the present invention consists of an isolated antigenic  Porphyromonas gingivalis  polypeptide or an antigenic fragment thereof, the polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ. ID. NO. 110; SEQ. ID. NO. 111; SEQ. ID. NO. 112; SEQ. ID. NO. 113; SEQ ID NO: 120; SEQ. ID. NO. 123; SEQ. ID. NO. 124; SEQ. ID. NO. 125; SEQ. ID. NO. 130; SEQ. ID. NO. 131; SEQ. ID. NO. 132; SEQ. ID. NO. 133; SEQ. ID. NO. 135; SEQ. ID. NO. 136; SEQ. ID. NO. 137; SEQ. ID. NO. 138; SEQ. ID. NO. 143; SEQ. ID. NO. 144; SEQ. ID. NO. 145; SEQ. ID. NO. 146; SEQ. ID. NO. 147; and SEQ. ID. NO. 148; 
         [0010]    or amino acid sequences at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ. ID. NO. 110; SEQ. ID. NO. 111; SEQ. ID. NO. 112; SEQ. ID. NO. 113; SEQ ID NO: 120; SEQ. ID. NO. 123; SEQ. ID. NO. 124; SEQ. ID. NO. 125; SEQ. ID. NO. 130; SEQ. ID. NO. 131; SEQ. ID. NO. 132; SEQ. ID. NO. 133; SEQ. ID. NO. 135; SEQ. ID. NO. 136; SEQ. ID. NO. 137; SEQ. ID. NO. 138; SEQ. ID. NO. 143; SEQ. ID. NO. 144; SEQ. ID. NO. 145; SEQ. ID. NO. 146; SEQ. ID. NO. 147; and SEQ. ID. NO. 148; 
         [0011]    or an amino acid sequence of at least 40 amino acids having a contiguous sequence of at least 40 amino acids identical to a contiguous amino acid sequence selected from the group consisting of: SEQ. ID. NO. 110; SEQ. ID. NO. 111; SEQ. ID. NO. 112; SEQ. ID. NO. 113; SEQ ID NO: 120; SEQ. ID. NO. 123; SEQ. ID. NO. 124; SEQ. ID. NO. 125; SEQ. ID. NO. 130; SEQ. ID. NO. 131; SEQ. ID. NO. 132; SEQ. ID. NO. 133; SEQ. ID. NO. 135; SEQ. ID. NO. 136; SEQ. ID. NO. 137; SEQ. ID. NO. 138; SEQ. ID. NO. 143; SEQ. ID. NO. 144; SEQ. ID. NO. 145; SEQ. ID. NO. 146; SEQ. ID. NO. 147; and SEQ. ID. NO. 148. 
         [0012]    As used herein % identity for polypeptides is to be calculated using the alignment algorithm of Needleman and Munsch (9) using a standard protein scoring matrix (Blosum 50). 
         [0013]    In a preferred embodiment the present invention provides a polypeptide or fragment thereof, the polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ. ID. NO. 110; SEQ. ID. NO. 111; SEQ. ID. NO. 112; SEQ. ID. NO. 113; SEQ ID NO: 120; SEQ. ID. NO. 123; SEQ. ID. NO. 124; SEQ. ID. NO. 125; SEQ. ID. NO. 130; SEQ. ID. NO. 131; SEQ. ID. NO. 132; SEQ. ID. NO. 133; SEQ. ID. NO. 135; SEQ. ID. NO. 136; SEQ. ID. NO. 137; SEQ. ID. NO. 138; SEQ. ID. NO. 143; SEQ. ID. NO. 144; SEQ. ID. NO. 145; SEQ. ID. NO. 146; SEQ. ID. NO. 147; and SEQ. ID. NO. 148. 
         [0014]    In a second aspect the present invention consists in an isolated DNA molecule, the DNA molecule comprising a nucleotide sequence which encodes the polypeptide of the first aspect of the present invention or a sequence which hybridises thereto under stringent conditions. 
         [0015]    It is preferred that the isolated DNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 36 to SEQ ID NO: 39; SEQ ID NO: 46; SEQ ID NO: 49 to SEQ ID NO: 51; SEQ ID NO: 56 to SEQ ID NO: 59; SEQ ID NO: 61 to SEQ ID NO:64; SEQ ID NO: 69 to SEQ ID NO: 74; SEQ ID NO: 3 to SEQ ID NO: 5; SEQ ID NO: 11; SEQ ID NO: 14 to SEQ ID NO: 16; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 24 to SEQ ID NO: 26; SEQ ID NO: 28 to SEQ ID NO: 33. 
         [0016]    In a third aspect the present invention consists in a recombinant expression vector comprising the DNA molecule of the second aspect of the present invention operably linked to a transcription regulatory element. 
         [0017]    The present invention also provides a cell comprising this recombinant expression vector. 
         [0018]    In a further aspect the present invention consists in a method for producing a  P. gingivalis  polypeptide comprising culturing the cell under conditions that permit expression of the polypeptide. 
         [0019]    In yet a further aspect the present invention provides a 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 present invention, or at least one DNA molecule of the second aspect of the present invention, or both,and a pharmaceutically acceptable carrier. It is preferred that the pharmaceutically acceptable carrier is an adjuvant. In other aspects the present invention provides methods of treating  P. gingivalis  infection in subject comprising the administration of the composition to the subject such that treatment of  P. gingivalis  infection occurs. The treatment may be prophylactic or therapeutic. 
         [0020]    In yet another aspect the present invention provides an antibody raised against a polypeptide of the first aspect the invention. The antibody may be polyclonal or monoclonal. The present invention also provides compositions including these antibodies. It is preferred that these compositions are adapted for oral use and may be, for example, dentrifices, mouthwashes, etc. 
         [0021]    In a still further aspect the present invention provides a nucleotide probe comprising at least 18 nucleotides and having a contiguous sequence of at least 18 nucleotides identical to a contiguous nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 5; SEQ ID NO: 11; SEQ ID NO: 14 to SEQ ID NO: 16; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 24 to SEQ ID NO: 26; SEQ ID NO: 28 to SEQ ID NO: 33; and sequences complementary thereto. It is preferred that the probe further comprises a detectable label. 
         [0022]    The present invention also provides a method for detecting the presence of  P. gingivalis  nucleic acid in a sample comprising:
       (a) contacting a sample with the nucleotide probe under conditions in which a hybrid can form between the probe and a  P. gingivalis  nucleic acid in the sample; and   (b) detecting the hybrid formed in step (a), wherein detection of a hybrid indicates the presence of a  P. gingivalis  nucleic acid in the sample.       
 
     
    
     DETAILED DESCRIPTION 
     Definitions 
       [0025]    A purified or isolated polypeptide or a substantially pure preparation of a polypeptide are used interchangeably herein and, as used herein, mean a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 mg of the polypeptide. 
         [0026]    A purified preparation of cells refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells. 
         [0027]    A purified or isolated or a substantially pure nucleic acid, e.g., a substantially pure DNA, (are terms used interchangeably herein) is a nucleic acid which is one or both of the following: not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5′ end and one at the 3′ end) in the naturally occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional  P. gingivalis  DNA sequence. 
         [0028]    A “contig” as used herein is a nucleic acid representing a continuous stretch of genomic sequence of an organism. 
         [0029]    An “open reading frame”, also referred to herein as ORF, is a region of nucleic acid which encodes a polypeptide. This region may represent a portion of a coding sequence or a total sequence and can be determined from a stop to stop codon or from a start to stop codon. 
         [0030]    As used herein, a “coding sequence” is a nucleic acid which is transcribed into messenger RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the five prime terminus and a translation stop code at the three prime terminus. A coding sequence can include but is not limited to messenger RNA synthetic DNA, and recombinant nucleic acid sequences. 
         [0031]    A “complement” of a nucleic acid as used herein refers to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence. 
         [0032]    A “gene product” is a protein or structural RNA which is specifically encoded by a gene. 
         [0033]    As used herein, the term “probe” refers to a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest. Probes are often associated with or capable of associating with a label. A label is a chemical moiety capable of detection. Typical labels comprise dyes, radioisotopes, luminescent and chemiluminescent moieties, fluorophores, enzymes, precipitating agents, amplification sequences, and the like. Similarly, a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest and immobilizes such molecule is referred herein as a “capture ligand”. Capture ligands are typically associated with or capable of associating with a support such as nitro-cellulose, glass, nylon membranes, beads, particles and the like. The specificity of hybridization is dependent on conditions such as the base pair composition of the nucleotides, and the temperature and salt concentration of the reaction. These conditions are readily discernible to one of ordinary skill in the art using routine experimentation. 
         [0034]    Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared×100. 
         [0035]    The terms peptides, proteins, and polypeptides are used interchangeably herein. 
         [0036]    An “immunogenic component” as used herein is a moiety, such as an  P. gingivalis  polypeptide, analog or fragment thereof, that is capable of eliciting a humoral and/or cellular immune response in a host animal. 
         [0037]    An “antigenic component” as used herein is a moiety, such as  P. gingivalis  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. 
         [0038]    As used herein, the term “cell-specific promoter” means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue. The term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well. 
         [0039]    As used herein, the term “control sequence” refers to a nucleic acid having a base sequence which is recognized by the host organism to effect the expression of encoded sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, terminators, and in some cases operators; in eukaryotes, generally such control sequences include promoters, terminators and in some instances, enhancers. The term control sequence is intended to include at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences. 
         [0040]    As used herein, the term “operably linked” refers to sequences joined or ligated to function in their intended manner. For example, a control sequence is operably linked to coding sequence by ligation in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence and host cell. 
         [0041]    A “sample” as used herein refers to a biological sample, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma. serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment. 
         [0042]    The practice of the invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present). The disclosure of these texts are incorporated herein by reference. 
       Pharmaceutically Acceptable Carriers 
       [0043]    The antibodies, polypeptides and DNA of the present invention can be included in compositions which include a carrier or diluent. These compositions include pharmaceutical compositions where the carrier or diluent will be pharmaceutically acceptable. Pharmaceutically acceptable carriers or diluents include those used in compositions suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. They are non-toxic to recipients at the dosages and concentrations employed. Representative examples of pharmaceutically acceptable carriers or diluents include, but are not limited to; water, isotonic solutions which are preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline) and can also contain one or more of, mannitol, lactose, trehalose, dextrose, glycerol, ethanol or polypeptides (such as human serum albumin). The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. 
         [0044]    As will be well understood by those skilled in the art alterations may be made to the amino acid sequences set out in the Sequence Listings. These alterations may be deletions, insertions, or substitutions of amino acid residues. The altered polypeptides can be either naturally occurring (that is to say, purified or isolated from a natural source) or synthetic (for example, by performing site-directed metagenesis on the encoding DNA). It is intended that such altered polypeptides which have at least 85%, preferably at least 95% 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. The level of % identity is to be calculated as set out above. 
         [0045]    Protein sequences are homologous if they are related by divergence from a common ancestor. Consequently, a species homologue of the protein will be the equivalent protein which occurs naturally in another species. Within any one species a homologue may exist as numerous allelic variants, and these will be considered homologues of the protein. Allelic variants and species homologues can be obtained by following standard techniques known to those skilled in the art. 
         [0046]    An allelic variant will be a variant that is naturally occurring within an individual organism. 
       Mutants, Variants and Homology—Nucleic Acids 
       [0047]    Mutant polynucleotides will possess one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed metagenesis on the DNA). It is thus apparent that polynucleotides of the invention can be either naturally occurring or recombinant (that is to say prepared using recombinant DNA techniques). 
         [0048]    An allelic variant will be a variant that is naturally occurring within an individual organism. 
         [0049]    Nucleotide sequences are homologous if they are related by divergence from a common ancestor. Consequently, a species homologue of the polynucleotide will be the equivalent polynucleotide which occurs naturally in another species. Within any one species a homologue may exist as numerous allelic variants, and these will be considered homologues of the polynucleotide. Allelic variants and species homologues can be obtained by following standard techniques known to those skilled in the art. 
       Antibody Production 
       [0050]    Antibodies, either polyclonal or monoclonal, which are specific for a polypeptide of the present invention can be produced by a person skilled in the art using standard techniques such as, but not limited to, those described by Harlow et al. Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press (1988), and D. Catty (editor), Antibodies: A Practical Approach, IRL Press (1988). 
         [0051]    Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of a protein. For the production of polyclonal antibodies, a number of host animals are acceptable for the generation of antibodies by immunization with one or more injections of a polypeptide preparation, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response in the host animal, depending on the host species, including but not limited to Freund&#39;s (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole lympet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. 
         [0052]    A monoclonal antibody to an epitope of a protein may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 493-497), and the more recent human B-cell hybridoma technique (Kesber et al. 1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al. 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). In addition, techniques developed for the production of “chimeric antibodies” by splicing the genes from antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity may be used (Mornison et al. 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al. 1984 Nature 312:604-608; Takeda et al. 1985 Nature 31:452-454). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce 4-specific single chain antibodies. 
         [0053]    Recombinant human or humanized versions of monoclonal antibodies are a preferred embodiment for human therapeutic applications. Humanized antibodies may be prepared according to procedures in the literature (e.g. Jones et al. 1986, Nature 321:522-25; Reichman et al. 1988 Nature 332:323-27; Verhoeyen et al. 1988, Science 239:1534-36). The recently described “gene conversion metagenesis” strategy for the production of humanized monoclonal antibody may also be employed in the production of humanized antibodies (Carter et al. 1992 Proc. Natl. Acad. Sci. U.S.A. 89:4285-89). Alternatively, techniques for generating the recombinant phase library of random combinations of heavy and light regions may be used to prepare recombinant antibodies (e.g. Huse et al. 1989 Science 246:1275-81). 
         [0054]    Antibody fragments which contain the idiotype of the molecule such as Fu F(ab1) and F(ab2) may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab) E2 fragment which can be produced by pepsin digestion of the intact antibody molecule; the Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragment, and the two Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Alternatively, Fab expression libraries may be constructed (Huse et al. 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragment with the desired specificity to a protein. 
       Adjuvants 
       [0055]    “Adjuvant” means a composition comprised of one or more substances that enhances the immunogenicity and efficacy of a vaccine composition. Non-limiting examples of suitable adjuvants include squalane and squalene (or other oils of animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil;  Corynebacterium -derived adjuvants such as  Corynebacterium parvum; Propionibacterium -derived adjuvants such as  Propionibacterium  acne;  Mycobacterium bovis  ( Bacillus Calmetic  and  Guerinn  or BCG); interleukins such as interleukin 2 and interleukin-12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOM adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A; dextran sulfate; DEAE-Dextran or DHAE-Dextran with aluminium phosphate; carboxypolymethylene such as Carbopol&#39; EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238); vaccinia or animal posvirus proteins; sub-viral particle adjuvants such as cholera toxin, or mixtures thereof. 
         [0056]    As used herein, stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO4 at 50° C.; (2) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt&#39;s solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1% SDS 
         [0057]    As will be understood the present invention includes within its scope DNA vaccination. Further information regarding DNA vaccination may be found in Donnelly et al, Journal of Immunological Methods 176(1994) 145-152, the disclosure of which is incorporated herein by reference. 
         [0058]    Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer, or group of elements or integers. 
       Preparation of the  P. Gingivalis  Library for Sequencing. 
       [0059]    To determine the DNA sequence of  P. gingivalis  genomic DNA was isolated from  P. gingivalis  strain W50 (ATCC 53978) essentially by the method described by Mamur J. (J. Mol. Biol. 3, 208-218, 1961). Cloning of DNA fragments was performed essentially as described by Fleischmann et al., (Science; 269, 496-512, 1995)(2). Briefly, purified genomic DNA from  P. gingivalis  was nebulized to fragment the DNA and was treated with Bal31 nuclease to create blunt ends then run twice through preparative 1% agarose gels. DNA fragments of 1.6-2.0 kb were excised from the gel and the DNA recovered. This DNA was then ligated to the vector pUC18 (SmaI digested and dephosphorylated; Pharmacia) and electrophoresed through a 1% preparative agarose gel. The fragment comprising linear vector plus one insert was excised, purified and this process repeated to reduce any vector without insert contamination. The recovered vector plus insert DNA was blunt-ended with T4 DNA polymerase, then a final ligation to produce circular DNA was performed. Aliquots of Epicurian Coli Electroporation-Competent Cells (Stratagene) were transformed with the ligated DNA and plated out on SOB agar antibiotic diffusion plates containing X-gal and incubated at 37° C. overnight. Colonies with inserts appeared white and those without inserts (vector alone) appeared blue. Plates were stored at 4° C. until the white clones were picked and expanded for the extraction of plasmid DNA for sequencing. 
       DNA Sequencing 
       [0060]    Plasmid DNA was prepared by picking bacterial colonies into 1.5 ml of LB, TB or SOB broth supplemented with 50-100 ug/ml Ampicillin in 96 deep well plates. Plasmid DNA was isolated using the QIAprep Spin or QIAprep 96 Turbo miniprep kits (QIAGEN GmbH, Germany). DNA was eluted into a 96 well gridded array and stored at −20C. 
         [0061]    Sequencing reactions were performed using ABI PRISM Dye Terminator and ABI PRISM BIGDye Terminator Cycle Sequencing Ready Reaction kits with AmpliTaq DNA polymerase FS (PE Applied Biosystems, Foster City, Calif.) using the M13 Universal forward and reverse sequencing primers. Sequence reactions were conducted on either a Perkin-Elmer GeneAmp 9700 (PE Applied Biosystems) or Hybaid PCR Express (Hybaid, UK) thermal cyclers. Sequencing reactions were analysed on ABI PRISM 377 DNA sequencers (PE Applied Biosystems). 
         [0062]    The sequences obtained are set out below. The relationship between these sequences is set out in Table 1. The initiation codon was calculated using a combination of sequence homology alignment (FASTA), signal sequence prediction (PSORT, SignalP) or ORF prediction (GeneMark). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Reference table indicating the relationships 
               
               
                 of each sequence ID to the selected proteins. 
               
             
          
           
               
                   
                 DNA 
                 Amino acid 
                   
                   
               
               
                   
                 sequence of 
                 sequence of 
                 DNA 
                 Amino acid 
               
               
                 Protein 
                 complete 
                 complete 
                 sequence of 
                 sequence of 
               
               
                 name 
                 ORF 
                 ORF 
                 protein 
                 protein 
               
               
                   
               
             
          
           
               
                 PG1 
                 1 
                 75 
                 34 
                 108 
               
               
                 PG10 
                 2 
                 76 
                 35 
                 109 
               
               
                 PG100 
                 3 
                 77 
                 36 
                 110 
               
               
                 PG102 
                 4 
                 78 
                 37, 38 
                 111, 112 
               
               
                 PG104 
                 5 
                 79 
                 39 
                 113 
               
               
                 PG11 
                 6 
                 80 
                 40 
                 114 
               
               
                 PG12 
                 7 
                 81 
                 41 
                 115 
               
               
                 PG13 
                 8 
                 82 
                 42 
                 116 
               
               
                 PG14 
                 9 
                 83 
                 43 
                 117 
               
               
                 PG2 
                 10 
                 84 
                 44, 45 
                 118, 119 
               
               
                 PG21 
                 11 
                 85 
                 46 
                 120 
               
               
                 PG22 
                 12 
                 86 
                 47 
                 121 
               
               
                 PG24 
                 13 
                 87 
                 48 
                 122 
               
               
                 PG25 
                 14 
                 88 
                 49 
                 123 
               
               
                 PG27 
                 15 
                 89 
                 50 
                 124 
               
               
                 PG28 
                 16 
                 90 
                 51 
                 125 
               
               
                 PG29 
                 17 
                 91 
                 52 
                 126 
               
               
                 PG3 
                 18 
                 92 
                 53 
                 127 
               
               
                 PG4 
                 19 
                 93 
                 54 
                 128 
               
               
                 PG5 
                 20 
                 94 
                 55 
                 129 
               
               
                 PG54 
                 21 
                 95 
                 56 
                 130 
               
               
                 PG57 
                 22 
                 96 
                 57, 58, 59 
                 131, 132, 133 
               
               
                 PG6 
                 23 
                 97 
                 60 
                 134 
               
               
                 PG68 
                 24 
                 98 
                 61, 62 
                 135, 136 
               
               
                 PG75 
                 25 
                 99 
                 63 
                 137 
               
               
                 PG76 
                 26 
                 100 
                 64 
                 138 
               
               
                 PG8 
                 27 
                 101 
                 65, 66, 67, 68 
                 139, 140, 141, 
               
               
                   
                   
                   
                   
                 142 
               
               
                 PG91 
                 28 
                 102 
                 69 
                 143 
               
               
                 PG94 
                 29 
                 103 
                 70 
                 144 
               
               
                 PG96 
                 30 
                 104 
                 71 
                 145 
               
               
                 PG97 
                 31 
                 105 
                 72 
                 146 
               
               
                 PG98 
                 32 
                 106 
                 73 
                 147 
               
               
                 PG99 
                 33 
                 107 
                 74 
                 148 
               
               
                   
               
             
          
         
       
     
       DNA Sequence Analysis 
       [0063]    DNA files in FASTA format were converted to GCG format files and imported into a database. The DNA files were translated into amino acid files using the program Flip obtained from ANGIS(Australian Genomic Information Service, University of Sydney, Australia). A series of bioinformatic analyses were performed on the proteins in order to select potential vaccine candidates. The programs used were FASTA homology searching (1), PSORT (2,3), SignalP (4), TopPred (5), and GeneMark (6). The proteins and their bioinformatic results were stored in the custom written database for search and retrieval of proteins with the desired characteristics 
         [0064]    The FASTA homology results for these proteins were then examined for any alignment with a protein suggesting surface location or vaccine efficacy. All proteins were searched for homology against a non-redundant bacterial protein database compiled by ANGIS using the FASTA algorithm. The settings used for the FASTA searches were Ktup=2, gap creation penalty=−12, gap extension penalty=−2, width for deriving alignment in opt=16 and the Blosum 50 scoring matrix. Individual FASTA search results were examined for significant homology by statistical probability and amino acid alignments. The results are set out in Table 2. 
         [0065]    Protein files were then trimmed to the first, second, third, fourth and fifth methionine residues using a protein trimming program (ANGIS). The trimmed proteins were then subjected to PSORT analysis for the detection of signal sequences and the prediction of cell location. Proteins exhibiting a PSORT probability of outer membrane &gt;0.8 were considered to indicate surface localisation. A second signal sequence detection program SignalP was also performed and, in certain instances, this program detected signals not identified with PSORT. All proteins identified by other methods were also analysed by PSORT and SignalP. Previously, the C-terminal amino acid of bacterial outer membrane proteins has been shown to be important for the assembly of the protein on the outer membrane (7). A typical structure definition for outer membrane proteins has been determined as the presence of a signal sequence at the N-terminus and a tyrosine or phenylalanine at the C-terminus. A number of the selected proteins exhibit this characteristic structure. The program TopPred was used to determine the presence and number of membrane spanning domains (MSDs) and the presence of such sequences indicates a preference to be attached to membranes such as the outer membrane. The results of PSORT, SignalP and TopPred analyses with the C-terminal amino acids of the selected proteins are set out in Table 3. 
         [0066]    The 70 amino acids from the C-terminus of a number of  P. gingivalis  outer membrane proteins share 50-100% protein sequence identity. These proteins included RGP1, RGP2, KGP, HagA, HagC, HagD, prtH and prtT. This conserved motif may be involved in the attachment or sorting of proteins to the outer membrane. The protein data set was searched using FASTA homology as described above and a number of novel proteins were identified which demonstrate similar motifs at their C-termini. The results are listed in Table 4 
         [0067]    The TonBIII box is a 30 amino acid motif present within TonB outer membrane receptors in a wide variety of bacteria. The TonBIII box of  P. gingivalis  (8) was used to search the protein data set for homology by FASTA as described above. Those proteins demonstrating significant homology are listed in Table 5. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 FASTA protein homology results of complete ORFs against a non-redundant protein database. 
               
             
          
           
               
                   
                   
                 Genbank 
                   
                 Length of 
                   
               
               
                 Protein 
                   
                 accession 
                 Length of 
                 
                   P. gingivalis 
                 
                 FASTA homology results 
               
             
          
           
               
                 name 
                 Homology description 
                 number 
                 homolog 
                 protein 
                 Identity % 
                 Overlap 
                 E value 
               
               
                   
               
               
                 PG1 
                 48 kD outer membrane protein,  Actinobacillus   
                 U24492 
                  449aa 
                  451aa 
                 32 
                  454aa 
                 1.40E−42 
               
               
                   
                 
                   pleuropneumoniae 
                 
               
               
                 PG2 
                 Outer membrane protein (susC),  Bacteroides   
                 L49338 
                 1038aa 
                 1017aa, 1014aa 
                 28 
                 1099aa 
                 4.60E−32 
               
               
                   
                 
                   thetaiotaomicron 
                 
               
               
                 PG3 
                 Outer membrane porin F adhesin,  Pseudomonas fluorescens   
                 U19743 
                  317aa 
                  223aa 
                 35 
                  187aa 
                 1.10E−10 
               
               
                 PG4 
                 Outer membrane protein A,  Escherichia fergusonii   
                 M63352 
                  243aa 
                  672aa 
                 48 
                  88aa 
                 4.10E−10 
               
               
                 PG5 
                 Adhesin protein (AdcA),  Streptococcus pneumoniae   
                 Z71552 
                  423aa 
                  315aa 
                 25 
                  279aa 
                 9.40E−15 
               
               
                 PG6 
                 Hemolysin A (phyA),  Prevotella melaninogenica   
                 U27587 
                  332aa 
                  324aa 
                 60 
                  306aa 
                 3.00E−74 
               
               
                 PG8 
                 Heme uptake protein A,  Bacteriodes fragilis   
                 X97122 
                  431aa 
                 598aa, 550aa, 
                 79 
                  417aa 
                 6.70E−121 
               
               
                   
                   
                   
                   
                 458aa, 426aa 
               
               
                 PG10 
                 Macrophage infectivity potentiator (MIP),  Legionella   
                 U92214 
                  234aa 
                  195aa 
                 50 
                  201aa 
                 4.70E−31 
               
               
                   
                   oakridgensis . 
               
               
                 PG11 
                 Haemagglutinin (phg),  Prevotella intermedia   
                 AF017417 
                  309aa 
                  313aa 
                 44 
                  309aa 
                 3.60E−44 
               
               
                 PG12 
                 Outer membrane lipoprotein,  Haemophilus influenzae   
                 M68502 
                  274aa 
                  271aa 
                 36 
                  254aa 
                 9.60E−27 
               
               
                 PG13 
                 Ferric receptor (cfrA),  Campylobacter coli   
                 U80812 
                  696aa 
                  757aa 
                 24 
                  625aa 
                 1.20E−18 
               
               
                 PG14 
                 36 kD antigen,  Helicobacter pylori   
                 U86610 
                  329aa 
                  331aa 
                 37 
                  326aa 
                 1.10E−35 
               
               
                 PG22 
                 Alpha-hemolysin gene,  Aeromonas hydrophila   
                 L36462 
                  85aa 
                  106aa 
                 57 
                  67aa 
                 2.60E−14 
               
               
                 PG24 
                 Putative hemolysin,  Streptococcus mutans   
                 AF051356 
                  445aa 
                  417aa 
                 29 
                  432aa 
                 1.80E−29 
               
               
                 PG25 
                 Cysteine protease,  Porphyromonas gingivalis   
                 U54691 
                 1723aa 
                  293aa 
                 42 
                  142aa 
                 1.10E−12 
               
               
                 PG27 
                 TonB linked adhesin,  Porphyromonas gingivalis   
                 Y07618 
                 1097aa 
                  312aa 
                 45 
                  360aa 
                 3.20E−41 
               
               
                 PG28 
                 Cysteine protease/hemagglutinin,  Porphyromonas gingivalis   
                 S75942 
                  886aa 
                  843aa 
                 35 
                  838aa 
                 7.00E−90 
               
               
                 PG54 
                 Protease 1,  Achromobacter lyticus   
                 J5128 
                  653aa 
                  940aa 
                 24 
                  695aa 
                 1.50E−22 
               
               
                 PG57 
                 Cysteine protease,  Porphyromonas gingivalis   
                 U68468 
                 1358aa 
                 924aa, 922aa, 
                 31 
                  742aa 
                 1.40E−23 
               
               
                   
                   
                   
                   
                 921aa 
               
               
                 PG75 
                 Class 3 outer membrane porin (porB),  Neisseria meningitidis   
                 U07191 
                  332aa 
                  391aa 
                 23 
                  239aa 
                 4.60E−01 
               
               
                 PG91 
                 Neuraminidase (nanH),  Bacteroides fragilis   
                 D28493 
                  544aa 
                  540aa 
                 24 
                  251aa 
                 1.60E+00 
               
               
                 PG94 
                 Arginyl endopeptidase,  Porphyromonas gingivalis   
                 D26470 
                  991aa 
                 1157aa 
                 24 
                  328aa 
                 7.60E−08 
               
               
                 PG21 
                 Surface antigen gene,  Methanosarcina mazei   
                 X84710 
                  783aa 
                  821aa 
                 37 
                  331aa 
                 6.20E−33 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Results of PSORT, SignalP and TopPred analysis of the proteins. The signal present column indicates the presence of a signal 
               
               
                 sequence detected with either PSORT or SignalP. The terms in parentheses indicates the type of signal sequence as determined by 
               
               
                 PSORT. The cell location &amp; probability values are generated by PSORT and represent the probability of the protein being in the cell 
               
               
                 compartments outer membrane (OM), inner membrane (IM), periplasmic space (PC) or cytoplasm (C). The number of transmembrane 
               
               
                 domains (TMDs) was determined by TopPred and does not include uncleavable signal sequences. 
               
             
          
           
               
                   
                 Protein 
                   
                   
                   
                 SignalP 
                 PSORT 
                   
                   
                   
               
               
                   
                 seqID 
                 Protein 
                   
                 Methionine in 
                 cleavage 
                 cleavage 
                 Cell Location &amp; probability 
                 C-terminal 
                 Number 
               
             
          
           
               
                 Protein name 
                 number 
                 Length 
                 Signal Present 
                 ORF 
                 site 
                 site 
                 OM 
                 IM 
                 PS 
                 C 
                 Amino Acid 
                 of TMD&#39;s 
               
               
                   
               
             
          
           
               
                 PG1 
                 386 
                  451aa 
                 Y 
                 1 
                 24 
                 34 
                 0 
                 0 
                 0 
                 0.22 
                 N 
                 0 
               
               
                 PG2 
                 424 
                 1017aa 
                 Y 
                 1 
                 20 
                 20 
                 0.94 
                 0 
                 0.33 
                 0 
                 F 
                 3 
               
               
                 PG2 
                 425 
                 1014aa 
                 Y 
                 2 
                 17 
                 17 
                 0.94 
                 0 
                 0.29 
                 0 
                 F 
                 3 
               
               
                 PG3 
                 434 
                  223aa 
                 Y (lipoprotein) 
                 1 
                 — 
                 18 
                 0.79 
                 0.76 
                 0 
                 0 
                 K 
                 3 
               
               
                 PG4 
                 447 
                  672aa 
                 Y (lipoprotein) 
                 1 
                 22 
                 22 
                 0.79 
                 0.7 
                 0 
                 0 
                 R 
                 0 
               
               
                 PG5 
                 458 
                  315aa 
                 Y 
                 1 
                 40 
                 35 
                 0 
                 0.25 
                 0 
                 0 
                 R 
                 0 
               
               
                 PG6 
                 475 
                  324aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.2 
                 S 
                 1 
               
               
                 PG8 
                 498 
                  598aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.22 
                 N 
                 0 
               
               
                 PG8 
                 499 
                  550aa 
                 N 
                 2 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.25 
                 N 
                 0 
               
               
                 PG8 
                 500 
                  458aa 
                 N 
                 3 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.34 
                 N 
                 0 
               
               
                 PG8 
                 501 
                  426aa 
                 N 
                 4 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.24 
                 N 
                 0 
               
               
                 PG10 
                 387 
                  195aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.11 
                 K 
                 0 
               
               
                 PG11 
                 400 
                  313aa 
                 Y 
                 1 
                 22 
                 26 
                 0.24 
                 0 
                 0.93 
                 0 
                 R 
                 1 
               
               
                 PG12 
                 411 
                  271aa 
                 Y (lipoprotein) 
                 3 
                 27 
                 29 
                 0.79 
                 0.7 
                 0 
                 0 
                 R 
                 0 
               
               
                 PG13 
                 419 
                  757aa 
                 Y 
                 1 
                 23 
                 25 
                 0.94 
                 0 
                 0.29 
                 0 
                 N 
                 0 
               
               
                 PG14 
                 420 
                  331aa 
                 Y (uncleavable) 
                 1 
                 35 
                 26 
                 0 
                 0.58 
                 0 
                 0 
                 K 
                 1 
               
               
                 PG21 
                 426 
                  821aa 
                 Y 
                 2 
                 24 
                 27 
                 0.34 
                 0 
                 0.37 
                 0 
                 G 
                 1 
               
               
                 PG22 
                 427 
                  106aa 
                 Y (uncleavable) 
                 1 
                 41 
                 41 
                 0 
                 0.29 
                 0 
                 0 
                 P 
                 0 
               
               
                 PG24 
                 429 
                  417aa 
                 Y 
                 1 
                 19 
                 19 
                 0 
                 0.44 
                 0 
                 0 
                 N 
                 3 
               
               
                 PG25 
                 430 
                  293aa 
                 Y 
                 1 
                 27 
                 28 
                 0.2 
                 0 
                 0.62 
                 0 
                 R 
                 0 
               
               
                 PG27 
                 431 
                  312aa 
                 N 
                 1 
                 — 
                 — 
                 0 
                 0 
                 0 
                 0.28 
                 Q 
                 1 
               
               
                 PG28 
                 432 
                  843aa 
                 Y 
                 1 
                 21 
                 21 
                 0.93 
                 0 
                 0.24 
                 0 
                 H 
                 1 
               
               
                 PG29 
                 433 
                  290aa 
                 Y 
                 1 
                 18 
                 16 
                 0.28 
                 0 
                 0.94 
                 0 
                 K 
                 1 
               
               
                 PG54 
                 463 
                  940aa 
                 Y 
                 1 
                 27 
                 20 
                 0.86 
                 0 
                 0.25 
                 0 
                 Q 
                 5 
               
               
                 PG57 
                 467 
                  925aa 
                 Y 
                 1 
                 28 
                 24 
                 0.53 
                 0 
                 0.2 
                 0 
                 P 
                 3 
               
               
                 PG57 
                 468 
                  922aa 
                 Y 
                 2 
                 25 
                 21 
                 0.53 
                 0 
                 0.2 
                 0 
                 P 
                 3 
               
               
                 PG57 
                 469 
                  921aa 
                 Y 
                 3 
                 24 
                 20 
                 0.53 
                 0 
                 0.2 
                 0 
                 P 
                 3 
               
               
                 PG68 
                 484 
                 1226aa 
                 Y 
                 1 
                 25 
                 25 
                 0.91 
                 0 
                 0.31 
                 0 
                 Y 
                 0 
               
               
                 PG68 
                 485 
                 1225aa 
                 Y 
                 2 
                 24 
                 24 
                 0.91 
                 0 
                 0.31 
                 0 
                 Y 
                 0 
               
               
                 PG75 
                 493 
                  391aa 
                 Y 
                 1 
                 26 
                 26 
                 0.94 
                 0 
                 0.3 
                 0 
                 H 
                 1 
               
               
                 PG76 
                 494 
                  446aa 
                 Y 
                 1 
                 21 
                 22 
                 0.94 
                 0 
                 0.32 
                 0 
                 V 
                 3 
               
               
                 PG91 
                 520 
                  540aa 
                 Y 
                 1 
                 25 
                 25 
                 0.85 
                 0 
                 0.30 
                 0 
                 E 
                 0 
               
               
                 PG94 
                 523 
                 1157aa 
                 Y 
                 1 
                 23 
                 28 
                 0.8 
                 0 
                 0.25 
                 0 
                 Q 
                 5 
               
               
                 PG96 
                 525 
                  563aa 
                 Y 
                 1 
                 23 
                 23 
                 0.40 
                 0 
                 0.33 
                 0 
                 K 
                 0 
               
               
                 PG97 
                 526 
                  437aa 
                 Y 
                 1 
                 23 
                 23 
                 0.32 
                 0 
                 0.65 
                 0 
                 Q 
                 0 
               
               
                 PG98 
                 527 
                  318aa 
                 Y (lipoprotein) 
                 1 
                 19 
                 19 
                 0.79 
                 0.7 
                 0 
                 0 
                 L 
                 1 
               
               
                 PG99 
                 528 
                  461aa 
                 Y (uncleavable) 
                 1 
                 22 
                 20 
                 0 
                 0 
                 0.3 
                 0 
                 R 
                 0 
               
               
                 PG100 
                 388 
                  279aa 
                 Y 
                 1 
                 20 
                 18 
                 0.26 
                 0 
                 0.54 
                 0 
                 I 
                 0 
               
               
                 PG102 
                 389 
                  562aa 
                 Y 
                 1 
                 29 
                 29 
                 0.19 
                 0 
                 0.4 
                 0 
                 S 
                 3 
               
               
                 PG102 
                 390 
                  558aa 
                 Y 
                 2 
                 25 
                 25 
                 0.26 
                 0 
                 0.46 
                 0 
                 S 
                 3 
               
               
                 PG104 
                 391 
                  391aa 
                 Y 
                 1 
                 17 
                 17 
                 0.62 
                 0 
                 0.22 
                   
                 R 
                 0 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Percentage identity and percentage similarity of various proteins with 
               
               
                 the 70 amino acids from the C-terminal of the  P. gingivalis  arginine 
               
               
                 protease 1 (RGP1), arginine protease 2 (RGP2), and the cysteine 
               
               
                 protease/hemagglutinin (prtT). 
               
             
          
           
               
                 Protein 
                 Percent identity 
                 Percent similarity 
               
             
          
           
               
                 name 
                 RGP1 
                 RGP2 
                 prtT 
                 RGP1 
                 RGP2 
                 prtT 
               
               
                   
               
             
          
           
               
                 PG21 
                 17 
                 29 
                 21 
                 40 
                 57 
                 49 
               
               
                 PG25 
                 43 
                 41 
                 9 
                 64 
                 73 
                 14 
               
               
                 PG27 
                 41 
                 33 
                 7 
                 73 
                 74 
                 11 
               
               
                 PG28 
                 21 
                 26 
                 34 
                 49 
                 57 
                 74 
               
               
                 PG54 
                 19 
                 13 
                 16 
                 40 
                 43 
                 33 
               
               
                 PG57 
                 11 
                 14 
                 19 
                 20 
                 24 
                 34 
               
               
                 PG91 
                 31 
                 21 
                 39 
                 57 
                 53 
                 74 
               
               
                 PG96 
                 0 
                 13 
                 20 
                 0 
                 24 
                 43 
               
               
                 PG97 
                 10 
                 26 
                 33 
                 14 
                 47 
                 61 
               
               
                 PG98 
                 16 
                 20 
                 0 
                 47 
                 54 
                 0 
               
               
                 PG99 
                 19 
                 0 
                 26 
                 41 
                 0 
                 54 
               
               
                 PG100 
                 20 
                 21 
                 24 
                 39 
                 57 
                 41 
               
               
                 PG102 
                 27 
                 20 
                 31 
                 50 
                 61 
                 61 
               
               
                 PG104 
                 16 
                 23 
                 26 
                 46 
                 44 
                 49 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Percentage identity and percentage similarity of various 
               
               
                 proteins with the TonBIII box of  P. gingivalis . 
               
             
          
           
               
                   
                 Protein name 
                 Percent identity 
                 Percent similarity 
               
               
                   
                   
               
               
                   
                 PG2 
                 46 
                 71 
               
               
                   
                 PG13 
                 57 
                 93 
               
               
                   
                   
               
             
          
         
       
     
       Cloning, Expression and Purification of Recombinant  P. Gingivalis  Genes. 
     PG1  
       [0068]    Oligonucleotides to the 5′ and 3′ regions of the deduced protein were used to amplify the gene of interest from a preparation of  P. gingivalis  W50 genomic DNA using the TaqPlus Precision PCR System (Stratagene) and a PTC-100 (MJ Research) thermal cycler or similar device. The 5′ oligonucleotide primer sequence was GCGCCATATGCTGGCCGAACCGGCC (SEQ ID NO: 149) the 3′ oligonucleotide primer sequence was GCGCCTCGAGTCAATTCATTTCCTTATAGAG (SEQ ID NO: 150). The PCR fragment was purified, digested with Nde I, Xho I restriction enzymes (Promega) and ligated into the corresponding sites of the plasmid pProEx-1 (Gibco-BRL) and transformed into  E. coli  ER1793 cells (a gift from Elizabeth Raleigh, New England Biolabs). A resulting clone expressing the correct insert was selected and induced with or without 0.1 mM IPTG (Promega) for expression of the recombinant protein. Expression of the recombinant protein was determined by SDS-PAGE analysis and Western Blot using the one of the rabbit antisera described above or an anti-hexahistidine antibody (Clontech) that detects the hexahistidine tag that was fused to the  P. gingivalis  recombinant protein. PG1 was purified by disruption of the  E. coli  cells by sonication in binding buffer (Novagen) and solubilisation by the addition of sarkosyl (N-Lauroyl sarcosine) to a 1% final concentration. There after the preparation was diluted to 0.1% sarkosyl in binding buffer, bound to a Nickel-nitrilotriacetic acid column (Ni—NTA; Qiagen), after washing bound proteins were eluted with 1M imidazole in elution buffer (Novagen) according to the Qiagen recommendations with 0.1% sarkosyl added to all buffers. Following purification samples were dialysed against 500 mM NaCl, 20 mM Tris, 0.1% sarkosyl at pH7.4 to remove the imidazole, concentrated as required and stored at 4° C. until used. Purity and antigenicity were assessed by SDS-PAGE and Western blot using selected antisera (from those described above) and the protein concentration was determined by the BCA assay (Pierce). 
       PG2  
       [0069]    The methods used for PG2 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGTATACATGAAAAGAATGACGC (SEQ ID NO: 151), the 3′ oligonucleotide primer sequence was CGCGAGATCTGAAAGACAACTGAATACC (SEQ ID NO: 152) and the PCR product was cloned into pGex-stop RBS(IV) (Patent application WO9619496, J C Cox, S E Edwards, I Frazer and E A Webb. Variants of human papilloma virus antigens) using the BstZ 171 and Bgl II restriction sites. 2% sarkosyl was used to solubilise PG2 and 8M urea was added to the solublisation buffer and to all other buffers. Urea was removed from the purified protein by sequential dialysis (4M then 2M then 1M then 0.5M then 0M urea all in 50 mM Tris, 500 mM NaCl, 0.1% sarkosyl, pH7.4). Purified protein was stored at 4° C. until required. 
       PG3  
       [0070]    The methods used for PG3 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGTATACATGAAGAAATCAAGTGTAG (SEQ ID NO: 153), the 3′ oligonucleotide primer sequence was GCGCAGATCTCTTCAGCGTACCTTGCTGTG (SEQ ID NO: 154) and DNA was amplified with Pfu DNA polymerase (Stratagene). The PCR product was cloned directly into pCR-Blunt and transformed into  E. coli  Top10F′(InVitrogen) before subcloning into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  BL21DE3 (Pharmacia Biotech). The following modifications were made to the purification of PG3 from the PG1 method. Cells expressing the recombinant protein were disrupted by sonication in binding buffer and the insoluble inclusion bodies concentrated by centrifugation. Inclusion bodies were then solubilised in 6M urea (Sigma) in binding buffer and eluted with 6M urea added to the elution buffer. In some instances 6M guanidine hydrochloride (Sigma) was used instead of urea for these steps. Urea (or guanidine hydrochloride when it was substituted) was removed from the purified protein by sequential dialysis against reducing levels of urea (3M then 1.5M then 0.5M then 0M urea all in 50 mM Tris, 500 mM NaCl, 8% glycerol, pH7.4). Purified protein was stored frozen at −80° C. until required. Protein concentration was determined by the Coomassie Plus protein assay (Pierce). 
       PG4  
       [0071]    The methods used for PG4 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CTTCTGTATACTTACAGCGGACATCATAAAATC (SEQ ID NO: 155), the 3′ oligonucleotide primer sequence was TTCCAGGAGGGTACCACGCAACTCTTCTTCGAT (SEQ ID NO: 156) and DNA was amplified with the Tth XL PCR kit (Perkin Elmer). The PCR product was cloned into the expression plasmid pgex-stop RBS(IV) using the Bst Z171 and Kpn I restriction sites and transformed into  E. coli  ER1793. 
       PG5  
       [0072]    The methods used for PG5 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was TTGCAACATATGATCAGAACGATACTTTCA (SEQ ID NO: 157) the 3′ oligonucleotide primer sequence was AGCAATCTCGAGCGGTTCATGAGCCAAAGC (SEQ ID NO: 158) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24 (Novagen) using the Nde I and Xho I restriction sites and transformed into  E. coli  BL21 (Pharmacia Biotech). Removal of urea was not proceeded past 1M urea as the protein was insoluble at lower concentrations of urea. Purified protein was stored at 4° C. until required. 
       PG6  
       [0073]    The methods used for PG6 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was TAAACATATGTGCCTCGAACCCATAATTGCTCCG (SEQ ID NO: 159), the 3′ oligonucleotide primer sequence was CGTCCGCGGAAGCTTTGATCGGCCATTGCTACT (SEQ ID NO: 160) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Nde I and Hind III restriction sites and transformed into  E. coli  BL21. 
       PG8  
       [0074]    The methods used for PG8 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGTATACATGGAGTTCAAGATTGTG (SEQ ID NO:161), the 3′ oligonucleotide primer sequence was CGCGAGATCTGTTTTCTGAAAGCTTTTC (SEQ ID NO: 162) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was cloned into the expression plasmid pProEx-1 using the Nde I and Xho I restriction sites and transformed into  E. coli  ER1793. 
       PG8A 
       [0075]    PG8A is a shortened version of PG8 and has the first 173 amino acids removed. The methods used for PG8A were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGTATACATGGAAAACTTAAAGAAC (SEQ ID NO: 163), the 3′ oligonucleotide primer sequence was CGCGAGATCTGTTTTCTGAAAGCTTTTC (SEQ ID NO: 164) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was cloned into the expression plasmid pgex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  ER1793. Prior to dialysis of the purified protein EDTA (Sigma) was added to a final concentration of 10 mM. 
       PG10  
       [0076]    The methods used for PG10 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGATATCATGGATAAAGTGAGCTATGC (SEQ ID NO:165), the 3′ oligonucleotide primer sequence was CGCGAGATCTTTTGTTGATACTCAATAATTC (SEQ ID NO: 166) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was digested with Eco RV and Bgl II and ligated into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  ER1793. 
       PG11  
       [0077]    The methods used for PG11 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGTATACATGAGAGCAAACATTTGGCAGATACTTTCCG (SEQ ID NO: 167), the 3′ oligonucleotide primer sequence was GCGCAGATCTGCGCAAGCGCAGTATATCGCC (SEQ ID NO: 168) and DNA was amplified with Tli DNA polymerase (Promega). The PCR product was cloned into pCR-Blunt and transformed into  E. coli  Top10F′ before subcloning into the expression plasmid pGex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  ER1793. PG11 was purified by solubilisation of  E. coli  cells with 2% sarkosyl in binding buffer (Qiagen) which was diluted to 0.1% sarkosyl in binding buffer, bound to a Nickel-nitrilotriacetic acid column (Ni—NTA; Qiagen), after washing bound proteins were eluted with 1M imidazole (0.7% CHAPS (Sigma) in elution buffer; Qiagen) according to the Qiagen recommendations. Following purification samples were dialysed against 500 mM NaCl, 20 mM Tris, 0.7% CHAPS, 20% glycerol (Sigma) at pH7.4 to remove the imidazole, concentrated as required and stored at 4° C. until used. 
       PG12  
       [0078]    The methods used for PG12 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGTATACATGAATAGCAGACATCTGACAATCACAATCATTGCCGG (SEQ ID NO: 169), the 3′ oligonucleotide primer sequence was GCGCAGATCTGCTGTTCTGTGAGTGCAGTTGTTTAAGTG (SEQ ID NO: 170) and DNA was amplified with Tli DNA polymerase. The PCR product was cloned into pCR-Blunt and transformed into  E. coli  Top10F′ cells before subcloning into the expression plasmid pgex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  BL21. Purification of the recombinant protein was essentially the same as PG11 except 0.5% DHPC (1,2-Diheptanoyl-sn-glycero-3-phosphocholine; Avanti) in 50 mM Tris, 50 mM NaCl, pH8.0 was used to solubilise the inclusion bodies instead of sarkosyl and the DHPC was diluted to 0.1% before addition to the Ni—NTA and 0.1% DHPC was added to all buffers. 
       PG13  
       [0079]    The methods used for PG13 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCCATATGCGGACAAAAACTATCTTTTTTGCG (SEQ ID NO:171), the 3′ oligonucleotide primer sequence was GCGCCTCGAGGTTGTTGAATCGAATCGCTATTTGAGC (SEQ ID NO: 172) and DNA was amplified with Tli DNA polymerase. The PCR product was cloned the expression plasmid pET24b using the Nde I and Xho I restriction sites and transformed into  E. coli  BL21. Purification of the recombinant protein was essentially the same as PG3 using 6M urea and 1% NOG (n-octyl glucoside; Sigma) was added to the dialysis buffer. Removal of urea was not proceeded past 2M urea as the protein was insoluble at lower concentrations of urea. Purified protein was stored at 4° C. until required. 
       PG14  
       [0080]    The methods used for PG12 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGGCGCCATGACGGACAACAAACAACGTAATATCG (SEQ ID NO: 173), the 3′ oligonucleotide primer sequence was GCGCCTCGAGTTACTTGCGTATGATCACGGACATACCC (SEQ ID NO: 174) and DNA was amplified with Tli DNA polymerase. The PCR product was cloned the expression plasmid pProEx-1 using the Ehe I and Xho I restriction sites and transformed into  E. coli  BL21. Purification of the recombinant protein was essentially the same as PG12. 
       PG22  
       [0081]    The methods used for PG22 were essentially the same as for PG1 with the following exceptions. The 5′ oligonucleotide primer sequence was CCCCGGATCCGATGCGACTGATCAAGGC (SEQ ID NO: 175), the 3′ oligonucleotide primer sequence was CCCCCTCGAGCGGAACGGGGTCATAGCC (SEQ ID NO: 176) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was cloned into the expression plasmid pET24b using the Bam HI and Xho I restriction sites and transformed into  E. coli  BL21DE3. Once PG22 was purified dialysis was performed in the same manner as for PG1 but in the presence of 1M imidazole. 
       PG24  
       [0082]    The methods used for PG24 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGGTATACATGAATTACCTGTACATAC (SEQ ID NO: 177), the 3′ oligonucleotide primer sequence was CGCGGGATCCGTTCGATTGGTCGTCGATGG (SEQ ID NO: 178) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was digested with Bst Z171 and Bam HI and ligated into the expression plasmid pgex-stop RBS(IV) using the Bst Z171 and Bgl II restriction sites and transformed into  E. coli  ER1793. Due to the low level of expression of PG24 purification was not proceeded with except on small scale. 
       PG24A 
       [0083]    A modified version of PG24 was also cloned and expressed. PG24A is the same as PG24 with the predicted N-terminal sequence removed. The methods used for PG24A were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was CGCGCATATGGAGATTGCTTTCCTTTCTTCG (SEQ ID NO: 179), the 3′ oligonucleotide primer sequence was CGCGCTCGAGTTAGTTCGATTGGTCGTCG (SEQ ID NO: 180) and DNA was amplified with the TaqPlus Precision PCR System. The PCR product was cloned into the expression plasmid pProEx-1 using the Nde I and Xho I restriction sites and transformed into  E. coli  ER1793. Purification of the recombinant protein was essentially the same as PG3 except 8M urea was used to solubilise the inclusion bodies and in the buffers used for the Ni—NTA column purification. Urea was removed by sequential dialysis (4M then 2M, then 1M then 0.5M then 0M urea all in 50 mM Tris, 500 mM NaCl, 8% glycerol, pH7.4). Purified protein was stored frozen at −80° C. until required. 
       PG29 
       [0084]    The methods used for PG29 were essentially the same as for PG3 with the following exceptions. The 5′ oligonucleotide primer sequence was GCGCGATATCGCTAGCATGAAAAAGCTATTTCTC (SEQ ID NO: 181), the 3′ oligonucleotide primer sequence was GCGCAGATCTCTCGAGTTTGCCATCGGATTGCGGATTG (SEQ ID NO: 182) and DNA was amplified with Pfu DNA polymerase being used. The PCR product was cloned into pCR-Blunt (InVitrogen) and transformed into  E. coli  Top10F′ before subcloning into the expression plasmid pGex-stop RBS(IV) using the EcoR V and Bgl II restriction sites and transformed into  E. coli  BL21. 6M urea was used throughout the purification process. 
       PG54  
       [0085]    The methods used for PG54 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was CGCTGAATTCCAGATTTCGTTCGGAGGGGAACCC (SEQ ID NO: 183), the 3′ oligonucleotide primer sequence was CTATGCGGCCGCCTGCTTCACGATCTTTTGGCTCA (SEQ ID NO: 184) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG57  
       [0086]    The methods used for PG57 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGGATCCCAAGAGATCTCAGGCATGAATGCA (SEQ ID NO: 185), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCGGCCTCTTTATCTCTACCTTTTC (SEQ ID NO: 186) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG68  
       [0087]    The methods used for PG68 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GCTTGCGGCCGCCCTTATGAAAGATTTGCAGAT (SEQ ID NO: 187), the 3′ oligonucleotide primer sequence was GGTGCTCGAGTATACTCAACAAGCACCTTATGCAC (SEQ ID NO: 188) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Not I and Xho I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG75  
       [0088]    The methods used for PG75 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCGGGATCCGCTCAGGAGCAACTGAATGTGGTA (SEQ ID NO: 189), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGTGGAACAAATTGCGCAATCCATC (SEQ ID NO: 190) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG76  
       [0089]    The methods used for PG76 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was AGCAGAATTCGGAAACGCACAGAGCTTTTGGGAA (SEQ ID NO: 191), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTACCTGCACCTTATGACTGAATAC (SEQ ID NO: 192) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG91  
       [0090]    The methods used for PG91 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCCAGACGATGGGAGGAGATGATGTC (SEQ ID NO: 193), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTTCCACGATGAGCTTCTCTACGAA (SEQ ID NO: 194) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG94  
       [0091]    The methods used for PG94 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCCGAGCTCCAAGAGGAAGGTATTTGGAATACC (SEQ ID NO: 195), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTTGTCCTACCACGATCATTTTCTT (SEQ ID NO: 196) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG96  
       [0092]    The methods used for PG96 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAGCTCCAAACGCAAATGCAAGCAGACCGA (SEQ ID NO: 197), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTTTGAGAATTTTCATTGTCTCACG (SEQ ID NO: 198) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Sac I and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG97  
       [0093]    The methods used for PG97 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCGGGATCCCAGTTTGTTCCGGCTCCCACCACA (SEQ ID NO: 199), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCTGTTTGATGAGCTTAGTGGTATA (SEQ ID NO: 200) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG98  
       [0094]    The methods used for PG98 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was AGCAGAATTCCAAGAAAGAGTCGATGAAAAAGTA (SEQ ID NO: 201), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTAGCTGTGTAACATTAAGTTTTTTATTGAT (SEQ ID NO: 202) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG99  
       [0095]    The methods used for PG99 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was TGCTGAATTCAAGGACAATTCTTCTTACAAACCT (SEQ ID NO: 203), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTCGAATCACGACTTTTCTCACAAA (SEQ ID NO: 204) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG100  
       [0096]    The methods used for PG100 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCAGAATTCCAGTCTTTGAGCACAATCAAAGTA (SEQ ID NO: 205), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTGATAGCCAGCTTGATGCTCTTAGC (SEQ ID NO: 206) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG102  
       [0097]    The methods used for PG102 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GGCCGAATTCCAGATGGATATTGGTGGAGACGAT (SEQ ID NO: 207), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTCTCTACAATGATTTTTTCCACGAA (SEQ ID NO: 208) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Eco RI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       PG104  
       [0098]    The methods used for PG104 were essentially the same as for PG3 with the following exceptions. The predicted N-terminal signal sequence was removed from the recombinant protein. The 5′ oligonucleotide primer sequence was GAACGGATCCAACGTGTCTGCTCAGTCACCCCGA (SEQ ID NO: 209), the 3′ oligonucleotide primer sequence was GAGTGCGGCCGCTTCTGAGCGATACTTTTGCACGTAT (SEQ ID NO: 210) and DNA was amplified with the Tth XL PCR kit. The PCR product was cloned into the expression plasmid pET24a using the Bam HI and Not I restriction sites and transformed into  E. coli  BL21DE3. Expression studies and immunoreactivity studies were carried out on whole  E. coli  lysates. Purification was not done for these studies. 
       Animal Antisera and Human Patient Sera. 
       [0099]    Various antisera were raised for detecting the expression and refolding of the recombinant  P. gingivalis  proteins. A whole cell antisera was raised by injecting New Zealand White rabbits with 3 doses of sonicated  P. gingivalis  (strain W50) containing approximately 2 mg of protein. The first dose was given in Freunds complete adjuvant (FCA) and the second and third doses were given in Freunds incomplete adjuvant (IFA) at 3 week intervals. Doses (1 ml) were given intramuscularly into the hind legs and rabbits bled 7 days after the last dose, the blood clotted and serum removed and stored at −20° C. until required. A second rabbit antisera was produced in a similar manner but using a sarkosyl insoluble fraction (each dose was 0.69 mg of protein) derived from  P. gingivalis  W50 according to the method of Doidg and Trust T. et al 1994 as the immunogen. A third rabbit antisera was produced in a similar manner to the first only the sarkosyl soluble fraction (1 mg of protein per dose) derived from  P. gingivalis  W50 cells according to the method of Doidg P. and Trust T J. (1994 Infect Immun 62:4526-33) was used as the immunogen. 
         [0100]    A “protected rat serum” pool was also used in these studies and was obtained from rats immunised with formalin killed whole  P. gingivalis  cells in FIA (strain ATCC 33277; 2 doses of 2×10 9  cells, 3 weeks apart). Rats were then challenged 2 weeks after their last dose with live  P. gingivalis  cells (strain 33277) given orally as previously described (Klaussen B. et al. 1991, Oral Microbiol Immunol 6:193-201) and the serum obtained from these rats 6 weeks after the final challenge inoculation at the time of sacrifice. 
         [0101]    Human sera were obtained from adult patients undergoing treatment or assessment for periodontitis at an outpatient clinic. These patients had at least 6 teeth with 6 mm attachment loss and had  P. gingivalis  present in their sub-gingival plaque as detected using a  P. gingivalis  specific DNA probe. Sera was pooled from these patients and compared to a pool of sera from periodontally healthy patients. 
       Immunization and Murine Lesion Model Protocols 
       [0102]    The mouse abscess model was used to assess the efficacy of immunising mice with recombinant  P. gingivalis  proteins in protecting mice from formation of a subcutaneous abscess. This model has been used by others as a predictor of potential vaccines against periodontal disease (Bird P S, et al. 1995 J. Periodontol. 66:351-362. BALB/c mice 6-8 weeks old were immunised by subcutaneously injecting them with 0.1 ml containing either 10 or 20 μg of recombinant  P. gingivalis  protein, 20 μg of  E. coli  lysate protein, 2×10 9  formalin killed cells of  P. gingivalis  strain 33277 emulsified in incomplete Freund&#39;s adjuvant (IFA; Sigma) on day 0. At day 21 mice were re-injected with the same dose and then bled 1 week later and evaluated for antibody levels. At day 35 mice all mice were challenged with approximately 2×10 9  cells of live  P. gingivalis  (ATCC 33277) by subcutaneous injection in the abdomen. Following challenge mice were monitored daily for weight loss and the size of the lesion measured for the next 10 days. Lesion sizes were measured by length and width and expressed as mm 2 . Groups were statistically analysed using a Kruskal-Wallis one-way ANOVA and were also individually examined using the unpaired t test or Mann-Whitney rank sum test using the Instat statistical package. 
         [0103]    FIG. 1 shows the results of one experiment at day 4 after challenge (lesions were at maximum size at this time point). Control mice immunised with  E. coli  lysate showed large lesions while mice immunised with killed cells of  P. gingivalis  strain 33277 were fully protected. This indicates that whole cells provide protection against  P. gingivalis  while  E. coli  protein immunised mice were not protected. Mice given the various PG recombinant proteins showed significant levels of protection for PG2, PG22, PG24 and PG29 (p&lt;0.05 unpaired t test) while PG8A was not quite significantly different (p=0.07) compared to the  E. coli  control group. 
         [0104]    FIG. 2 shows the results of a separate experiment using combinations of recombinant proteins. Mice given PG1+PG2 showed a significant level of protection compared to control mice give  E. coli  lysate (p&lt;0.026 unpaired t test). 
       Immunoscreening 
       [0105]    Cloned candidates were cultured in 15 ml of Terrific broth, induced with IPTG and sampled at 4 h post-induction. One ml of culture was removed, pelleted and the cells resuspended in a volume of PBS determined by dividing the OD A 600nm  of the culture by 8. An aliquot of lysate (100 μl) was added to 100 μl of 2× sample reducing buffer (125 mM Tris pH 6.8, 20% glycerol, 4% SDS, 80 mM DTT, 0.03% bromophenol blue) and boiled for 10 min. SDS-PAGE was performed according to the method of Laemmli UK. 1970 (Nature 227:680-685) using 4-20% 1.0 mm Tris-Glycine gels (Novex) according to the manufacturers recommendations. Proteins were transferred onto Hybond-C Extra nitrocellulose membranes (Amersham) by transblotting and the membranes were then blocked for 2 h at room temperature (RT) in 5% skim milk in 20 mM Tris, 0.5M NaCl, 0.05% Tween-20, pH 7.5 (TTBS). 
         [0106]    Immunoscreening was performed separately with the rabbit anti- P. gingivalis  whole cell serum, the rat protective serum, a pool of human periodontal patients serum, and in many cases an anti-T7-Tag antibody HRP conjugate (Novagen). Prior to use, the rabbit, rat and human sera were diluted 1/5000, 1/1000 and 1/500 respectively in 5% skim milk in TTBS and absorbed with 100 μl (for the rabbit serum) or 250 μl (for the rat and human sera)  E. coli  extract (20 mg/ml; Promega) for 6 h at RT. 
         [0107]    Membranes were incubated overnight at RT with the absorbed antisera, or for 1 hr at RT with 1/5000 diluted anti-T7-Tag conjugate. Following 3×10 min washes with TTBS, HRP-conjugated anti-rabbit (Silenus), anti-mouse (Silenus) or anti-human (KPL) antibody, diluted 1/5000 in 5% skim milk in TTBS, was added for 1 h at RT. Membranes were washed as before, prior to addition of TMB membrane peroxidase substrate (KPL) for detection of immunoreactive proteins. Results of reactivity for the recombinant  P. gingivalis  proteins is shown in Table 7. 
         [0108]    In addition some of the sera (pooled sera diluted 1/1000) from the mice immunised with  P. gingivalis  recombinant proteins (prior to challenge) were analysed for their reactivity against Western blots of whole native W50  P. gingivalis  proteins using similar techniques as those outlined above. PG2, PG8A, PG29 and PG3 all showed bands at a similar molecular weight to that of the recombinant PG protein in the native W50 blot. This indicates that PG proteins are expressed in the W50 strain and that the recombinant proteins have at least some identical immunogenicity to the native proteins. 
         [0000]    m-RNA Analysis 
       Hot Phenol RNA Extraction 
       [0109]      P. gingivalis  W50 cells (150 ml culture) were grown anaerobically to mid log phase (OD A 600 =0.18) mixed with 50% glycerol and stored at −70° C. until RNA extraction. Cells were pelleted by centrifugation at 6000 g, and resuspended in 8 ml ASE (20 mM NaOAc, 0.5% SDS, 1 mM EDTA). An equal volume of 20 mM NaOAc(pH 4.5)-saturated phenol was added and mixed by shaking for 30 seconds, incubated at 65° C. for 5 minutes, followed by a further 5 second shaking and repeated incubation. After cooling, 2 ml chloroform was added and mixed by shaking for 5 seconds, and the mixture spun at 10000 g for 10 minutes at 4° C. The top aqueous phase was transferred and re-extracted by repeating the phenol and chloroform steps. The aqueous phase was transferred again and 100 U RNase inhibitor (RNAsin; Promega) were added. RNA was precipitated with 3 volumes 100% ethanol at −20° C. overnight. The RNA precipitate was recovered by centrifugation at 10000 g at 4° C. for 15 minutes, then washed with 100% ethanol, dried and resuspended in 600 μl sterile, deionised, dH 2 O with 1 μl of fresh RNase inhibitor. RNA was aliquoted and stored at −70° C. The RNA concentration was determined spectrophotometrically. A formaldehyde RNA gel confirmed RNA integrity (Sambrook J. et al. 1989, Molecular Cloning. A laboratory manual. Cold Spring Laboratory Press, New York. 2nd Edition). 
       RT-PCR 
       [0110]    The isolated RNA was used as a template for Reverse Transcription (RT) to produce cDNA. Varying RNA concentrations were used for the RT as each RNA transcript was potentially present at different levels. Subsequent amplification of the cDNA was performed using Polymerase Chain Reaction (PCR). RT-PCR was performed using GeneAmp® RNA PCR Kit (Perkin Elmer) according to the manufacturer&#39;s protocol with the following exception to the PCR; 35 cycles were performed as follows: Melt phase 95° C. for 30 seconds, Anneal phase varied between 50-60° C. for 30 seconds, Extension phase 72° C. for 1 minute. Amplification was performed in a PTC-100 Programable Thermal Controller (MJ Research Inc.). As a control to demonstrate that the amplified product did not arise from contaminating DNA, Reverse Transcriptase (RTase) was omitted from a parallel tube. The PCR products were examined against DNA markers (GIBCO 1 kB ladder) on a 1% agarose gel stained with ethidium bromide. 
         [0111]    RT-PCR results are shown in Table 6 using the oligonucleotide primers as used in “Cloning, expression and purification of recombinant  P. gingivalis  genes” section described above,except for the following changes. For PG1 the 3′ reverse primer used was GCGCCTCGAGATTCATTTCCTTATAGAG (SEQ ID NO: 211), for PG4 the 5′ forward primer was CTTCTTGTCGACTACAGCGGACATCATAAAATC (SEQ ID NO: 212) and the 3′ reverse primer was TTCCACCTCGAGTTAACGCAACTCTTCTTCGAT (SEQ ID NO: 213), for PG6 the 5′ forward primer was TAAAGAATTCTGCCTCGAACCCATAATTGCTCCG (SEQ ID NO: 214), for PG10 the 5′ forward primer was CGCGCATATGGATAAAGTGAGCTATGC (SEQ ID NO: 215) and the 3′ reverse primer was CGCGCTCGAGTTTGTTGATACTCAATAATTC (SEQ ID NO: 216), for PG13 the 5′ forward primer was GCCCGGCGCCATGCGGACAAAAACTATCTTTTTTGCG (SEQ ID NO: 217) and the 3′ reverse primer was GCCCGGCGCCTTAGTTGTTGAATCGAATCGCTATTTGAGC (SEQ ID NO: 218). Amplification of  P. gingivalis  transcripts is a likely indication that RNA for a specific candidate is present and that the protein is produced. However, where there is no amplification achieved this does not indicate that this gene is never transcribed and may be the result of the culture conditions or the state of the cells when harvested. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Expression of PG m-RNA with in vitro grown  P. gingivalis  W50. The 
               
               
                 symbols are + band visible on agarose gel, − no band present 
               
               
                 on agarose gel, ND not detected. 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Approx. 
                 Expected 
               
               
                   
                 RNA 
                 Annealing 
                   
                 PCR 
                 fragment 
                 fragment 
               
               
                 PG # 
                 μg 
                 temp. ° C. 
                 RT-PCR 
                 (−RT) 
                 size bp 
                 size bp 
               
               
                   
               
             
          
           
               
                  1 
                 0.15 
                 55 
                 + 
                 − 
                 1300 
                 1362 
               
               
                  2 
                 1.0 
                 50 
                 + 
                 − 
                 3200 
                 3051 
               
               
                  3 
                 0.15 
                 60 
                 + 
                 − 
                 720 
                 690 
               
               
                  4 
                 2.9 
                 55 
                 − 
                 − 
                 N.D. 
                 2000 
               
               
                  5 
                 0.02 
                 50 
                 + 
                 − 
                 1000 
                 947 
               
               
                  6 
                 1.0 
                 55 
                 + 
                 − 
                 1000 
                 972 
               
               
                  8A 
                 0.15 
                 50 
                 + 
                 − 
                 1200 
                 1278 
               
               
                 10 
                 0.15 
                 55 
                 + 
                 − 
                 590 
                 585 
               
               
                 11 
                 0.10 
                 60 
                 + 
                 − 
                 960 
                 942 
               
               
                 12 
                 0.02 
                 60 
                 + 
                 − 
                 880 
                 831 
               
               
                 13 
                 1.0 
                 50 
                 + 
                 − 
                 2150 
                 2274 
               
               
                 14 
                 0.15 
                 60 
                 + 
                 − 
                 1050 
                 996 
               
               
                 22 
                 1.0 
                 60 
                 − 
                 − 
                 N.D. 
                 228 
               
               
                 24 
                 1.0 
                 55 
                 + 
                 + 
                 1150 
                 1194 
               
               
                 29 
                 0.15 
                 60 
                 + 
                 − 
                 880 
                 885 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Immunoblot results of proteins expressed in  E. coli  against rabbit, rat and 
               
               
                 human antisera. Deduced MW was calculated from amino acid sequence 
               
               
                 of the  P. gingivalis  proteins, some of which had their N-terminal signal 
               
               
                 sequences removed. Apparent MW was determined from SDS-PAGE gels. 
               
               
                 The N- and C-terminal tags add approximately 2.5 KDa to the deduced 
               
               
                 MW of the recombinant proteins. The symbols are + positive, 
               
               
                 − negative, +/− weak positive, ND not done. 
               
             
          
           
               
                 Protein 
                 Deduced MW 
                 Apparent 
                 Antisera reactivity 
               
             
          
           
               
                 number 
                 (KDa) 
                 MW (KDa) 
                 T7 
                 Rabbit 
                 Rat 
                 Human 
               
               
                   
               
             
          
           
               
                 PG1 
                 47.5 
                 63 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG2 
                 112.4 
                 125.7 
                 ND 
                 + 
                 − 
                 − 
               
               
                 PG3 
                 22.6 
                 18.3 
                 ND 
                 − a   
                 − 
                 − 
               
               
                 PG4 
                 75 
                 90.6 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG5 
                 34.9 
                 43.8 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG6 
                 36.7 
                 47.1 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG8 
                 67.5 
                 63.1 
                 ND 
                 − b   
                 − 
                 − 
               
               
                 PG8A 
                 47.7 
                 90.6 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG10 
                 21.3 
                 25.5 
                 ND 
                 + 
                 − 
                 + 
               
               
                 PG11 
                 36.2 
                 42.4 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG12 
                 30.7 
                 30.6 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG13 
                 84.5 
                 101 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG14 
                 36 
                 42.4 
                 ND 
                 − 
                 + 
                 + 
               
               
                 PG22 
                 8.6 
                 11.1 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG24A 
                 47 
                 63.1 
                 ND 
                 − 
                 − 
                 − 
               
               
                 PG29 
                 31.1 
                 40.9 
                 ND 
                 + 
                 + 
                 + 
               
               
                 PG54 
                 101.4 
                 46.7 
                 + 
                 + 
                 − 
                 − 
               
               
                 PG57 
                 100 
                 134.5 
                 + 
                 + 
                 + 
                 + 
               
               
                 PG68 
                 133.3 
                 30.7 
                 + 
                 − 
                 + 
                 + 
               
               
                 PG75 
                 40.7 
                 46.7 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG76 
                 48.1 
                 55.6 
                 + 
                 − 
                 − 
                 + 
               
               
                 PG91 
                 57.2 
                 85.7 
                 + 
                 + 
                 + 
                 + 
               
               
                 PG96 
                 59.3 
                 70.3 
                 + 
                 + 
                 + 
                 + 
               
               
                 PG97 
                 44.4 
                 57.5 
                 + 
                 − 
                 + 
                 + 
               
               
                 PG98 
                 33.3 
                 36 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG99 
                 40.7 
                 55.6 
                 + 
                 − 
                 + 
                 + 
               
               
                 PG100 
                 29.6 
                 10.8 
                 + 
                 − 
                 − 
                 − 
               
               
                 PG102 
                 59.3 
                 70.3 
                 + 
                 − 
                 − 
                 + 
               
               
                 PG104 
                 40.7 
                 57.5 
                 + 
                 − 
                 − 
                 + 
               
               
                   
               
               
                   a Positive reaction detected with the rabbit antiserum to sarkosyl insoluble  P. gingivalis  antigen. 
               
               
                   b Purified protein demonstrated weak positive reaction with the rabbit antiserum to whole  P. gingivalis . 
               
             
          
         
       
     
         [0112]    It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. 
       REFERENCES  
       [0000]    
       
         1. Lipman D J, Pearson W R. 1985. Rapid and sensitive protein similarity searches. Science 277:1435-1441. 
         2. Horton, P. and Nakai, K. (1996). A probabilistic classification system for predicting the cellular localization sites of proteins. Intellig. Syst. Mol. Biol. 4: 109-115. 
         3. Nakai K, Kanehisa M. 1991. Expert systems for predicting protein localization sites in Gram-negative bacteria. Proteins: Structure, Function, and Genetics 11:95-110. 
         4. Nielsen H, Engelbrecht J, Brunak S and von Heijne G. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10, 1-6. 
         5. Claros M G and G von Heijne. (1994). TopPred II: an improved software for membrane protein structure predictions. Comput. Appl. Biosci. 10: 685-686. 
         6. Borodovsky M, Rudd K E, and E V Koonin. (1994). Intrinsic and extrinsic approaches for detecting genes in a bacterial genome. Nucleic Acids Res. 22:4756-4767. 
         7. Struvye M, Moons M, Tommassen J. 1991. Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein J. Mol. Biol. 218:141-148. 
         8. Aduse-Opoku J, Slaney J M, Rangarajan M, Muir J, Young K A, Curtis M A. 1997. The T1a receptor protein of  Porphyromonas gingivalis  W50: a homolog of the RI precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin. J. Bacteriol. 179:4778-4788. 
         9. Needleman S B, Munsch C D. 1970. Ageneral method applicable to the search of similarity in the amino acid sequence of two proteins. J. Molec. Biol. 48: 443-453.