Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Testing efficacy or toxicity of a compound or composition
Reexamination Certificate
1998-07-07
2003-03-04
Swartz, Rodney P (Department: 1645)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Testing efficacy or toxicity of a compound or composition
C424S184100, C424S190100, C424S234100, C514S900000, C514S901000, C514S902000, C530S300000, C530S350000, C536S023100, C536S023700
Reexamination Certificate
active
06528038
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to oral compositions and immunogenic compositions for use in the suppression of the pathogenic effects of the bacterium
Porphyromonas gingivalis
associated with periodontal disease and cardiovascular disease. It also relates to diagnostic tests for the presence of
Porphyromonas gingivalis
in subgingival plaque samples and specific antibodies against
P. gingivalis
antigens. The compositions comprise proteins, peptides or oligopeptides or peptide chimeras of specific antigens of
Porphyromonas gingivalis
. Also disclosed are methods for preparing the antigens, peptide components and peptide chimeras using recombinant DNA and/or biochemical techniques. Related thereto, disclosed are the DNA sequences encoding the specific antigens, and recombinant vectors useful in directing the expression of antigen constructs containing major epitopes. Also disclosed are host cells transformed with such recombinant vectors. The proteins, peptides, oligopeptides and peptide chimeras are useful as immunogens in formulations for use in raising an immune response and can be used to generate protein-specific and peptide-specific antisera useful for passive immunization and as reagents for diagnostic assays. The nucleotide sequences disclosed provide for the synthesis of corresponding oligonucleotides which can be used as reagents in diagnostic assays directed to the detection of
P. gingivalis
genetic material and incorporated into expression vectors for use as genetic vaccine formulations.
BACKGROUND OF THE INVENTION
Periodontal diseases are bacterial-associated inflammatory diseases of the supporting tissues of the teeth and range from the relatively mild form of gingivitis, the non-specific, reversible inflammation of gingival tissue to the more aggressive forms of periodontitis which are characterised by the destruction of the tooth's supporting structures. Periodontitis is associated with a subgingival infection of a consortium of specific Gram-negative bacteria that leads to the destruction of the periodontium and is a major public health problem. One bacterium that has attracted considerable interest is
Porphyromonas gingivalis
as the recovery of this microorganism from adult periodontitis lesions can be up to 50% of the subgingival anaerobically cultivable flora, whereas
P. gingivalis
is rarely recovered, and then in low numbers, from healthy sites. A proportional increase in the level of
P. gingivalis
in subgingival plaque has been associated with an increased severity of periodontitis and eradication of the microorganism from the cultivable subgingival microbial population is accompanied by resolution of the disease. The progression of periodontitis lesions in non-human primates has been demonstrated with the subgingival implantation of
P. gingivalis
. These findings in both animals and humans suggest a major role for
P. gingivalis
in the development of adult periodontitis. The presence of
P. gingivalis
in atheromatous plaques has also been associated with the development of cardiovascular disease.
P. gingivalis
is a black-pigmented, anaerobic, proteolytic Gram-negative rod that obtains energy from the metabolism of specific amino acids. The microorganisms has an absolute growth requirement for iron, preferentially in the form of heme or its Fe(III) oxidation product hemin and when grown under conditions of excess hemin is highly virulent in experimental animals. A number of virulence factors have been implicated in the pathogenicity of
P. gingivalis
including the capsule, adhesins, cytotoxins and extracellular hydrolytic enzymes. In order to develop an efficacious and safe vaccine to prevent
P. gingivalis
colonisation it is necessary to identify protein antigens that are involved in virulence that have utility as immunogens to generate neutralising antibodies.
SUMMARY OF THE INVENTION
The present inventors purified and characterised four major
P. gingivalis
antigens using serum from a healthy subject that harboured
P. gingivalis
in subgingival plaque as shown by DNA probe analysis. The antigens (Ag1, Ag2, Ag3 and Ag4) are listed below.
Putative
A
M
r
Function
Internal Amino acid sequence
Ag1
32 kDa
Haeme
DLENKGEATLLVTFGSSYKAPRETYAKIEK
receptor
TFAAAYPDQR (SEQ ID NO: 1)
Ag2
46 kDa
Fimbrial
DNPDENPLEGDITQTHTEKYVLAED (SEQ ID NO: 2)
protein
Ag3
70 kDa
DnaK
DVLLLDVTPLSLGIETMGGVMTYLIDANTT
homologue
IPKLK (SEQ ID NO: 3)
Ag4
10 kDa
S-layer protein
VYNASISAVGNTSAIDPVVQIIHHN (SEQ ID NO: 4)
(N-terminal sequence)
Accordingly in a first aspect the present invention consists in a composition for use in raising an immune response directed against
Porphyromonas gingivalis
, the composition including a suitable adjuvant and/or acceptable carrier and one substantially purified
P. gingivalis
immunogen, the immunogen being selected from the group consisting of Antigen 1, Antigen 2, Antigen 3, Antigen 4 and epitope containing fragments thereof. Optionally, the composition may further include at least one additional purified
P. gingivalis
immunogen, the immunogen being selected from the group consisting of Antigen 1, Antigen 2, Antigen 3, Antigen 4 and epitope containing fragments thereof.
In a second aspect, the present invention consists in a substantially purified
P. gingivalis
antigen or epitope containing fragment thereof, wherein antigen has an internal amino acid sequence:
DLENKGEATLLVTFGSSYKAPRETYAKIEKTFAAAYPDQR (SEQ ID NO:1). It is preferred that the antigen has an amino acid sequence as shown in
FIG. 1
(SEQ ID NOS:5 and 6).
In a third aspect, the present invention consists in a substantially purified
P. gingivalis
antigen or epitope containing fragment thereof, wherein antigen has an internal amino acid sequence:
DNPDENPLEGDITQTHTEKYVLAED (SEQ ID NO:2).
In a fourth aspect, the present invention consists in a substantially purified
P. gingivalis
antigen or epitope containing fragment thereof, wherein antigen has an internal amino acid sequence:
DVLLLDVTPLSLGIETMGGVMTYLIDANTTIPKLK (SEQ ID NO:3). It is preferred that the antigen includes an amino acid sequence encoded by the open reading frame of the clone deposited with AGAL under accession No. NM 97/04974 which hybridises with degenerate probes corresponding to the amino acid sequence DVLLLDVTPLSLGIETMGGVMTYLIDANTTIPKLK (SEQ ID NO:3).
In a fourth aspect, the present invention consists in a substantially purified
P. gingivalis
antigen antigen or epitope containing fragment thereof, wherein antigen has an internal amino acid sequence: VYNASISAVGNTSAIDPVVQIIHHN (SEQ ID NO:4).
In other aspects, the present invention consists in nucleotide sequences encoding Ag1, Ag2, Ag3 and Ag4 and probes which hybridise to these sequences.
The nucleotide sequence encoding Ag1 and deduced amino acid sequence of the haeme receptor protein Ag1 is shown in FIG.
1
. The disclosure of the nucleotide sequence includes within its scope degeneracy equivalents and subsequences coding for amino acid sequences corresponding to antigenic determinants of
P. gingivalis
W50.
A clone containing nucleotide sequence from Ag3, DnaK clone #6, has been deposited under the terms of the Budapest Treaty with Australian Government Analytical Laboratories, 1 Suakin Street, Pymble, NSW Australia on Mar. 25, 1997 and has been accorded accession No. NM 97/04974. Accordingly further nucleotide sequence for this antigen can be obtained by accessing this deposit. Access to this deposit is available under the terms and conditions of the Budapest Treaty. Where applicable access to this deposit is to be limited to independent experts (EPC Rule 28(4). AU Reg. 3.25(3)).
In another aspect the present invention consists in antibodies raised against the antigens of the present invention.
Antibodies against the antigens can be used in oral compositions such as toothpaste and mouthwash to neutralise the antigens and thus prevent disease. Antigen-specific antibodies can also be used for the early detection of
P. gingivalis
in subgingival plaque samples by a diagnostic assay. A vaccine based on these antigens and
Hendtlass Anne
Reynolds Eric Charles
Slakeski Nada
Swartz Rodney P
The University of Melbourne
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