Diagnostics and treatments of periodontal disease

Details Diagnostics and treatments of periodontal disease Diagnostics and treatments of periodontal disease

1998-09-15

2003-01-28

Kunz, Gary L. (Department: 1647)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

C424S185100, C424S190100, C424S193100, C424S197110

Reexamination Certificate

active

06511666

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the PrtR-PrtK cell surface protein of
Porphyromonas gingivalis
and in particular a multimeric cell associated protein complex comprising the PrtR and PrtK proteins. The invention also relates to pharmaceutical compositions and associated agents based on said complex for the detection, prevention and treatment of Periodontal disease associated with
P. gingivalis.
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
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.
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. In particular, proteases have received a great deal of attention for their ability to degrade a broad range of host proteins including structural proteins and others involved in defence. The proteins that have been shown to be substrates for
P. gingivalis
proteolytic activity include collagen types I and IV, fibronectin, fibrinogen, laminin, complement and plasma clotting cascade proteins, &agr;
1
-antitrypsin, &agr;
2
-macroglobulin, antichymotrypsin, antithrombin III, antiplasmin, cystatin C, IgG and IgA. The major proteolytic activities associated with this organism have been defined by substrate specificity and are “trypsin-like”, that is cleavage on the carboxyl side of arginyl and lysyl residues and collagenolytic although other minor activities have been reported.
P. gingivalis
trypsin-like proteolytic activity has been shown to degrade complement, generating biologically active C5a, impair the phagocytic and other functions of neutrophils by modifying surface receptors, and abrogate the clotting potential of fibrinogen prolonging plasma clotting time. The trypsin-like proteolytic activity of
P. gingivalis
also generates Fc fragments from human IgG1 stimulating the release of pro-inflammatory cytokines from mononuclear cells and is associated with vascular disruption and enhanced vascular permeation through the activation of the kallikrein-kinin cascade.
P. gingivalis
spontaneous mutants with reduced trypsin-like activity as well as wild-type cells treated with the trypsin-like protease inhibitor N-p-tosyl-L-lysine chloromethyl ketone are avirulent in animal models. Further, it has been shown that
P. gingivalis
grown under controlled, haemin-excess conditions expressed more trypsin-like and less collagenolytic activity and were more virulent in mice relative to cells grown under haemin-limited but otherwise identical conditions. The increased expression of the trypsin-like activity by the more virulent
P. gingivalis
has led to the speculation that the trypsin-like proteolytic activity may be the major determinant for infection or disease. However, the cell-associated trypsin-like proteolytic activities of
P. gingivalis
have not been characterised to date.
There has been considerable endeavour to purify and characterise the trypsin-like proteases of
P. gingivalis
from cell-free culture fluids. Chen et al, (1992) [J Biol Chem 267:18896-18901] have purified and characterised a 50 kDa arginine-specific, thiol protease from the culture fluid of
P. gingivalis
H66 designated Arg-gingipain. A similar arginine-specific thiol protease has been disclosed in JP 07135973 and the amino acid sequence disclosed in WO 9507286 and in Kirszbaum et al, 1995 [Biochem Biophys Res Comm 207:424-431]. Pike et al (1994) [J Biol Chem 269:406-411] have characterised a 60 kDa lysine-specific cysteine proteinase from the culture fluid of
P. gingivalis
H66 designated Lys-gingipain and the partial gene sequence for this enzyme was disclosed in WO 9511298 and fully disclosed in WO 9617936. However, prior to the development of the present invention it was unknown that there existed on the cell surface of
P. gingivalis a
300 kda complex of arginine-specific and lysine-specific proteases both containing adhesin domains. The 300 kDa complex has been designated the PrtR-PrtK complex. The presence of the PrtR-PrtK cell surface complex exhibiting both arginine- and lysine-specific proteolytic activity together with adhesin activity was previously unknown. Furthermore, the new PrtR-PrtK complex of the present invention is expressed on the cell surface, is a major virulence-associated factor and contains unique epitopes not displayed on the individual domains. The previously disclosed arginine-specific and lysine-specific thiol proteases, as discussed, do not exhibit any of these features and have proven of limited application to date. However, the aforementioned features have rendered the PrtR-PrtK complex of the invention ideal for development of diagnostic and immunoprophylactic products. The PrtR-PrtK cell surface complex is accordingly of particular interest for diagnostics and neutralisation by passive immunity through oral compositions containing neutralising antibodies and by vaccine development. In particular for the development of an intra-oral recombinant bacterial vaccine, where the recombinant bacterium expressing an inactivated PrtR-PrtK is a genetically engineered commensal inhabitant of the oral cavity.
SUMMARY OF THE INVENTION
Accordingly in a first aspect the present invention consists in a substantially purified antigenic complex for use in raising an antibody response directed against
Porphyromonas gingivalis,
the complex comprising at least one multimeric protein complex of arginine-specific and lysine-specific thiol endopeptidases each containing at least one adhesin domain, the complex having a molecular weight of greater than about 200 kDa.
In the context of this disclosure, the terms “adhesin” and “hemagglutinin” may be considered to be synonymous.
In a preferred form of the present invention the multimeric protein complex is associated with virulent strains of
Porphyromonas gingivalis,
preferably has a molecular weight of about 294 to about 323 kDa and is preferably derived from
P. gingivalis
W50.
It is also preferred that the multimeric protein complex is composed of 9 proteins. These 9 proteins preferably have the following N-terminal sequences:
DVYTDHGDLYNTPVRML (SEQ ID NO: 1)
YTPVEEKQNGRMIVIVAKKYEGD (SEQ ID NO: 2)
SGQAEIVLEAHDVWNDGSGYQILLDADHDQYGQVIPSDTHFL (SEQ ID NO: 3)
PQSVWIERTVDLPAGTKYVAFR (SEQ ID NO: 4)
ANEAKVVLAADNVWGDNTGYQFLLDA (SEQ ID NO: 5)
ANEAKVVLAADNVWGD

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