Diagnostics and treatments of periodontal disease

a multi-meric cell and disease technology, applied in the field of diagnosis and treatment of periodontal disease, can solve the problems of limited application to date, major public health problems, and cell-associated trypsin-like proteolytic activities of i>p. gingivalis /i> have not been characterised to date,

Inactive Publication Date: 2011-09-01
UNIVERSITY OF MELBOURNE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018]In a fifth aspect the present invention consists in a method of reducing the prospect of P. gingivalis infection in an individual and / or severity of disease, the method comprising administering to the individual an amount of the composition of the second aspect of the present invention effective to induce an immune response in the individual directed against P. gingivalis.
[0020]In another aspect, the present invention is directed to novel DNA sequences involving PrtR-PrtK constructs and vectors including plasmid DNA, and viral DNA such as human viruses, animal viruses, insect viruses, or bacteriophages which can be used to direct the expression of PrtR-PrtK protein in appropriate host cells from which the expressed protein may be purified. Another aspect of the present invention provides methods for molecular cloning of the genes encoding the PrtR-PrtK complex. The nucleic acid sequences of the present invention can be used in molecular diagnostic assays for P. gingivalis genetic material through nucleic acid hybridization, and including the synthesis of PrtR-PrtK sequence-specific oligonucleotides for use as primers and / or probes in amplifying, and detecting amplified, nucleic acids. Additionally, PrtR-PrtK complex can be used as an immunogen in prophylactic and / or therapeutic vaccine formulations against pathogenic strains of P. gingivalis, whether the immunogen is chemically synthesized, purified from P. gingivalis, or purified from a recombinant expression vector system. Alternatively, the genes encoding PrtR-PrtK may be incorporated into a bacterial or viral vaccine comprising recombinant bacteria or virus which is engineered to produce PrtR-PrtK by itself, or in combination with immunogenic epitopes of other pathogenic microorganisms. In addition, the genes encoding PrtR-PrtK operatively linked to one or more regulatory elements, can be introduced directly into humans to express the PrtR-PrtK to elicit a protective immune response. A vaccine can also be based upon a recombinant component of a mutated PrtR-PrtK incorporated into an appropriate vector and expressed in a suitable transformed host (eg. E. coli, Bacillus subtilis, Saccharomyces cerevisiae, COS cells, CHO cells and HeLa cells) containing the vector. The vaccine can be based on an intra-oral recombinant bacterial vaccine, where the recombinant bacterium expressing an inactivated PrtR-PrtK is a commensal inhabitant of the oral cavity. Unlike whole P. gingivalis cells or other previously prepared antigens based on fimbriae or the capsule the PrtR-PrtK complex of the invention or component parts thereof are safe and effective antigens for the preparation of a vaccine for the prevention of P. gingivalis-associated periodontal disease. The invention therefore provides a range of recombinant products based on the PrtR-PrtK complex.

Problems solved by technology

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.
However, the cell-associated trypsin-like proteolytic activities of P. gingivalis have not been characterised to date.
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.

Method used

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  • Diagnostics and treatments of periodontal disease
  • Diagnostics and treatments of periodontal disease
  • Diagnostics and treatments of periodontal disease

Examples

Experimental program
Comparison scheme
Effect test

example 1

(1) Preparation of Antigen

A. Anion Exchange and Affinity Chromatography

[0076]P. gingivalis W50 was grown anaerobically at 37° C. on lysed horse blood agar and in modified BM media containing 1 μg / ml hemin. Bacteria were maintained on lysed horse blood plates by routine passage (P. gingivalis was grown to late logarithmic phase and the cells harvested by centrifugation (5,000×g, 20 min, 4° C.) and then resuspended in 160 ml TC buffer (20 mM Tris-HCl pH 7.4 and 5 mM CaCl2) containing 50 mM NaCl and subjected to mild sonication using a Branson Sonifier 250 with an output control of 3 and a 50% duty cycle for 15 min at 4° C. The sonicate was centrifuged (100,000×g, 30 min, 4° C.) and the supernatant filtered (0.22 μm) prior to anion-exchange FPLC. The sonicate was applied to an anion-exchange column (Hiload XK 16 / 10 Q Sepharose, Pharmacia-LKB) cooled to 4° C., in multiple injections using a 50 ml superloop (Pharmacia-LKB). The sample was eluted using a linear gradient from 0-100% buffer...

example 2

[0092]Methods and compounds for vaccine formulations related to PrtR-PrtK.

[0093]This embodiment of the present invention is to provide PrtR-PrtK protein to be used in as an immunogen in a prophylactic and / or therapeutic vaccine for active immunization to protect against or treat infections caused by P. gingivalis. For vaccine purposes, an antigen of P. gingivalis comprising a bacterial protein should be immunogenic, and induce functional antibodies directed to one or more surface-exposed epitopes on intact bacteria, wherein the epitope(s) are conserved amongst strains of P. gingivalis.

[0094]In one illustration of the PrtR-PrtK protein having the properties desirable of a vaccine antigen, the protein was purified from P. gingivalis using the method described herein in Example 1. Mice were immunized with the purified inactivated PrtR-PrtK protein (25 μg) with adjuvant (20 ug of QS21) two times at four week intervals. The purified PrtR-PrtK was inactivated by air oxidation. Blood from...

example 3

[0099]Protective Efficacy of Immunisation with the PrtR-PrtK Complex in an Animal Model.

[0100]Various preparations of purified P. gingivalis proteins were tested in the mouse abscess model. This model is loosely based on the methods described by Kesavalu et al (1992) [Infect Immun 60:1455-1464]. A typical experiment is outlined below. Briefly BALB / c mice were obtained from ARC (Perth, Australia) and were immunised subcutaneously in the scruff of the neck with the preparations and doses according to Table 5 before challenge with live P. gingivalis strain W50, which was given at 10 weeks of age. Mice were given 2 doses of vaccine at 4 and 1 weeks before challenge. Formalin killed P. gingivalis W50 cells were prepared by incubating an aliquot of cells in 0.5% (vol / vol) of buffered formal saline overnight at 4° C. The chloroform extract of P. gingivalis was prepared as detailed in Example 2. Purification of PrtR-PrtK complex was performed as detailed in Example 1. The PrtR-PrtK domains ...

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Abstract

This invention relates to the PrtR-PrtK cell surface protein of Porphyromonas gingivalis and in particular a multimeric cell association protein complex comprising the PrtR and PrtK proteins. Accordingly the invention provides a substantially purified antigenic complex for use in raising an antibody response directed against Porphyromonas gingivalis. The complex comprises 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. 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.

Description

CROSS REFERENCES TO RELATED APPLICATIONS[0001]This application is a continuation of application Ser. No. 11 / 654,512, filed Jan. 18, 2007, which is a continuation of Ser. No. 10 / 229,066, filed Aug. 28, 2002, which is a continuation of application Ser. No. 09 / 066,330, filed Sep. 15, 1998, and claims priority to Australian Application No. PN 6275, filed Oct. 30, 1995, which is a 371 of PCT / AU96 / 00673, filed Oct. 30, 1996, the entire contents of each of which is hereby incorporated by reference in this application.FIELD OF THE INVENTION[0002]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[0003]Periodontal diseases are bact...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/40C12N9/76A61K31/00A61K38/00A61K39/00A61K39/02A61K39/395A61K39/40A61P1/00A61P1/02A61P37/00A61P37/04C07K2/00C07K16/12C12N9/52
CPCA61K8/64A61K39/00G01N33/56955C07K16/1203C12N9/52A61Q11/00A61K38/00A61P1/00A61P1/02A61P37/00A61P37/04C12N9/6427C07K16/12
Inventor REYNOLDS, ERIC CHARLESBHOGAL, PETER SINGHSLAKESKI, NADA
Owner UNIVERSITY OF MELBOURNE
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