KR100843443B1 - Vaccine for periodontal disease - Google Patents

Abstract

The present invention provides novel bacterial isolates identified by 16S rRNA DNA, polynucleotide sequences contained in these bacteria, polypeptides encoded by these polynucleotide sequences, and the bacteria, polynucleotides or polypeptides that cause periodontal disease in companion animals. It relates to a vaccine comprising a. The present invention also provides a method for treating and preventing periodontal disease, a kit for detecting and treating periodontal disease, and a kit for detecting and preventing periodontal disease. Also provided are methods for assessing the efficacy of a vaccine against periodontal disease in an animal.

16S rRNA DNA, periodontal disease, pets, vaccines.

Description

Vaccine for periodontal disease {VACCINE FOR PERIODONTAL DISEASE}

The present invention provides novel bacterial isolates identified by their 16S rRNA DNAs, polynucleotide sequences contained therein, polypeptides encoded by such polynucleotide sequences, that cause periodontal disease in companion animals, or are inactivated or It relates to a vaccine comprising such attenuated bacterial isolates, polynucleotides or polypeptides. Also provided are methods of treating and preventing periodontal disease and kits for detecting, treating and preventing periodontal disease. Also provided is a method of evaluating the efficacy of a vaccine against periodontal disease in an animal.

Most experimental data on periodontal disease is based on the study of humans or bacteria isolated from humans. Relatively little is known about periodontal disease in pets, especially non-human animals such as dogs and cats.

Periodontal disease includes a group of infections associated with the supportive tissue of the tooth. These range from moderate and reversible inflammation of the gingival (gum) to severity, to chronic destruction of the periodontal tissue (gingival, periodontal ligament and alveolar bone) with consequent detachment of the tooth.

From a microbiological standpoint, some features of the disease are interesting. Bacterial etiology is complicated by the variety of organisms responsible for initiation and progression of the disease in humans. Many, if not all, of these organisms may also be present in periodontal healthy individuals, and may coexist harmoniously with the host. Thus, disease episodes may occur as a result of shifts in ecological balance between bacteria and host factors, for example as a result of changes in the absolute or relative numbers of certain organisms, changes in etiology potentials, or regulation of certain host factors. have. The local environment exerts various unique pressures on the microflora that make up the gingival tooth surface and subgingival fissures (the channel between the gingiva and the root that deepens into the periodontal pocket) as the disease progresses.

Both calcified hard tissues of teeth and epithelial cells of gingiva are available for colonization. The tissue is exposed to host saliva secretion and gingival sulcus (serum exudate), both of which contain molecules that interact directly with bacteria and change prevailing environmental conditions. It is also known that successful colonization of the tooth and subgingival areas in humans must coexist with many other species of bacteria (greater than 600) that inhabit these areas. Thus, the study of the pathogenesis of periodontal disease in humans is complicated by the ecological complexity of the microenvironment.

It will be sufficient to mention that the classification of various findings of periodontal disease in humans is constantly changing, the disease varies in severity, rate of progression and number of teeth affected, and that different age groups may be susceptible after the development of major teeth. . The nature of the pathogenic agent varies not only in the disease entity but also in human patients and even among different disease sites in the patient. In general, however, the severe form of the disease is associated with the number of Gram-negative anaerobic bacteria. Most of the evidence in this group in humans points to a pathogenic role for Porphyromonas (formerly Bacteroides ) Porphyromonas gingivalis . As alone or as a mixed infection with other bacteria, the absence of bacterial species and possibly the presence of such organisms that work in concert with specific immunological responses in the host appear to be essential for disease activity.

Colonization of the oral cavity requires that bacteria first enter the mouth and then be located and attached to the available surface. Host factors that function to prevent bacterial colonization include the mechanical shear force of tongue movement along with saliva and gingival fluid flow. Thus, successful oral colonization species have various contributors to overcome host protection mechanisms. Adherent plaque biofilms that accumulate continuously on the hard and soft tissues of the mouth are dynamic systems involved in a variety of microbial species. In humans, blood. Genjivalis is a late or second colony species of the oral cavity that typically requires the preceding organisms to create the necessary environmental conditions.

blood. It is believed that the initial influx of zinbaris into the human oral cavity is caused by transmission from infected individuals. Thus, other vectors also appear to be functional. The study indicates that the individual is colonized by a single (or at least dominant) genotype regardless of the colonization site or clinical condition. In contrast, strains of many different clone origins exist in different individuals. This is blood. It supports the notion that seriously bilivalis is essentially an opportunistic pathogen with virulence that is not limited to specific clone types.

Human oral blood. It provides a variety of surfaces to which the ear ballis can be attached. There are mineralized hard tissues of the teeth along with the mucosal surfaces, including the gingiva, jaw and tongue.

As described above, very much is known about periodontal disease in humans, while little is known about the disease in companion animals. Fournier, D. et al., “ Porphyromonas gulae sp. nov., an Anaerobic, Gram- negative, Coccibacillus from the Gingival Sulcus of Various Animal Hosts ", International Journal of a Systematic and Evolutionary Microbiology (2001), 51, 1179-1189] , the strain P designated ATCC 57100. gulrae (P . Gulae) there is a several strains isolated from various animal hosts, including the species (nov.) is described. the authors blood displacement of the balise the strains for the animal biotype blood Pseudomonas formate fatigue distinct seriously Bali Su species It is hypothesized that no vaccine is useful for the treatment of periodontal disease in companion animals [Hirasawa and Takada, in " Porphyromonas gingivicanis sp. nov. and Porphyromonas crevioricanis sp. nov., Isolated from Beagles ", International Journal of Systemic Bacteriology, pp. 637-640, (1994), describes two bacterial species isolated from Beagle's gingival sulcus. These species are described in US Pat. No. 5,710,039 and No. 5,563,063, the authors do not imply any use of this species in vaccines to treat periodontal disease, the international application PCT / AU98 / 01023, publication number WO 99/29870. Bally's polypeptides and nucleotides have been described, however, there is no evidence for vaccines effective in the prevention of periodontal disease in companion animals, although a great deal of information is known about human disease, Very little has been achieved by prevention or treatment.

There remains a need for safe and effective vaccines for the treatment and prevention of periodontal disease in companion animals.

Genco et al. Trends in Microbiology 6: 444-449, 1998, describes a rat model for the examination of Porphyromonas gingivicanis -mediated periodontal disease. Grecca et al. J. Endodontics 27: 610, 2001, describes a radiological assessment of perioral restoration after root canal therapeutic treatment of dog teeth with induced perioral periodontitis.

Prior to the present invention, there was no animal model available to evaluate the efficacy of a vaccine against one or more periodontal pathogenic bacteria in a defined and quantitative manner.

Summary of the Invention

The present invention provides isolated stained anaerobic bacteria having 16S rRNA DNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 86-94, but not a strain of Porphyromonas gingivalis, designated as dog 20B.

In one embodiment, the bacterium is Porphyromonas oyster B43, p. P. cansulci B46, p. P. circumdentaria B52, p. Whale B69, blood. Sukhumvitaria B97, p. P. cangingivalis B98, p. P. salivosa B104, oh. O. denticanis B106 and p. P. endodontalis B114 is selected from the group consisting of, except that the bacterium is not a strain of Porphyromonas gingivalis designated dog 20B.

In another embodiment, the present invention causes periodontal disease in a companion animal directly or in combination with other pathogenic agents, and comprises periodontal disease in a companion animal comprising an inactivated or attenuated immunologically effective amount of one or more bacteria It may be used to prepare a vaccine for the treatment or prophylaxis of blood, provided that it is designated as ATCC 51700. Provides isolated stained anaerobic bacteria that are not strains of ula. Preferably, the bacterium has a 16S rRNA DNA sequence that is at least about 95% homologous to any sequence shown in SEQ ID NOs: 86-94. More preferably, the bacterium has a 16S rRNA DNA sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 86-94.

In another embodiment, the present invention induces periodontal disease in companion animals, directly or in combination with other pathogenic agents, and includes an isolated polypeptide that is immunologically effective as a vaccine for the prevention or treatment of periodontal disease in companion animals. And the polypeptide may be used to prepare a vaccine for the treatment or prevention of periodontal disease in a companion animal, wherein the polypeptide is encoded by a polynucleotide molecule isolated from bacteria, provided that the blood is designated ATCC 51700. Provides isolated stained anaerobic bacteria that are not strains of ula. Preferably, the bacterium has a 16S rRNA DNA sequence that is at least about 95% homologous to any of the sequences shown in SEQ ID NOs: 86-94. More preferably, the bacterium has a 16S rRNA DNA sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 86-94.

In a further embodiment, the present invention is directed to causing periodontal disease in companion animals, directly or in combination with other pathogenic agents, and to isolate an immunologically effective polypeptide as a vaccine for the prevention or treatment of periodontal disease in companion animals. Polynucleotide molecules, wherein the polynucleotide molecules are isolated from bacteria and can be used to prepare a vaccine for the treatment or prevention of periodontal disease in a companion animal, provided that the blood is designated ATCC 51700. Provides isolated stained anaerobic bacteria that are not strains of ula. Preferably, the bacterium has a 16S rRNA DNA sequence that is at least about 95% homologous to any of the sequences shown in SEQ ID NOs: 86-94. More preferably, the bacterium has a 16S rRNA DNA sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 86-94.

The companion animal is preferably a dog or cat.

In another aspect, the present invention provides Porphyromonas gul B43, p. Kansul B46, p. Sukhumvitaria B52, p. Whale B69, blood. Sukhumvitaria B97, p. Kanjinjivalis B98, p. Salibosa B104, oh. Dentikanis B106 and p. A blood selected from the group consisting of bacteria having the identified characteristics of Endodontalis B114, but designated ATCC 51700. Provided is an isolated polynucleotide molecule comprising a nucleotide sequence isolated from a bacterium that is not a strain of a native species (nov.).

In one embodiment, the isolated polynucleotide molecule is porphyromonas dulla B43, p. Kansul B46, p. Sukhumvitaria B52, p. Whale B69, blood. Sukhumvitaria B97, p. Kanjinjivalis B98, p. Salibosa B104, oh. Dentikanis B106 and p. It is isolated from bacteria selected from the group consisting of endodontalis B114.

In another embodiment, the isolated polynucleotide encodes a polypeptide.

In another embodiment, the isolated polynucleotides encode ribosomal RNA or carrier RNA.

In another further embodiment, the invention includes any nucleotide sequence selected from the group consisting of SEQ ID NOs: 86-94 and homologues having at least 95% homology therewith, provided that the nucleotide sequence is designated as ATCC 51700. It provides an isolated polynucleotide molecule that is not a strain of the ola species (nov.).

Preferably, the isolated polynucleotide molecule is selected from the group consisting of SEQ ID NOs: 95-102 and 111-119 ( fimA or oprF , respectively) encoding polypeptides immunologically effective as vaccines for the prevention or treatment of periodontal disease in companion animals. Any nucleotide sequence or complement thereof.

The isolated polynucleotide molecule can also be any nucleotide sequence shown in SEQ ID NOs: 95-102 and 111-119, which encodes an immunologically effective polypeptide as a vaccine for the prevention or treatment of periodontal disease in a companion animal, at least 95% thereof. It is preferred to include homologues having homology, or complements thereto.

In a further embodiment, the isolated polynucleotide molecule is any nucleotide sequence shown in SEQ ID NOs: 95-102 and 111-119 encoding an immunologically effective polypeptide as a vaccine for the prevention or treatment of periodontal disease in a companion animal or Fragments or variants thereof, or complements thereto.

In another further embodiment, an isolated polynucleotide molecule comprises a nucleotide sequence that hybridizes under any stringent conditions to any of the sequences shown in SEQ ID NOs: 95-102 and 111-119, or complements thereto. Preferably, the isolated polynucleotide sequence is a sequence of fimA wherein said sequence is selected from any of the sequences shown in SEQ ID NOs: 95-102 , fragments or variants thereof having at least about 95%, 98%, or 99% sequence identity thereto. It includes. In addition, an isolated polynucleotide molecule includes a sequence of oprF wherein said sequence is selected from any of the sequences shown in SEQ ID NOs: 95-102 , fragments or variants thereof having at least about 95%, 98%, or 99% sequence identity thereto. It is desirable to.

Preferably, fragments or variants of the polynucleotide molecules according to the invention are at least about 98% homologous thereto.

In another embodiment, the present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence hybridized with fimA under high stringency conditions, or a complement thereof, selected from any of the sequences shown in SEQ ID NOs: 95-102 .

In another embodiment, the present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence that hybridizes with oprF under high stringency conditions, or a complement thereof, selected from any of the sequences shown in SEQ ID NOs: 111-119 .

The present invention also provides high stringency conditions with DNA molecules having a nucleotide sequence encoding a polypeptide having a sequence of about 10 or more consecutive amino acids of any polypeptide encoded by any of the nucleotide sequences of SEQ ID NOs: 95-102 and 109-119. An isolated polynucleotide molecule is provided comprising a nucleotide sequence of about 30 nucleotides, or complements thereof, hybridized below. Preferably, the isolated polynucleotide molecule has a nucleotide sequence encoding a polypeptide having a sequence of about 30 or more contiguous amino acids of any polypeptide encoded by any of the nucleotide sequences of SEQ ID NOs: 95-102 and 111-119. At least about 90 nucleotides, or complements thereof, that hybridize under very stringent conditions to the DNA molecule.

In another aspect, the present invention provides an isolated polynucleotide according to the present invention operably linked to a heterologous promoter. Isolated polynucleotides may further comprise an origin of replication active in prokaryotic or eukaryotic cells.

In another aspect, the invention provides a recombinant expression vector comprising a polynucleotide selected from the group consisting of any nucleotide sequence of SEQ ID NOs: 95-102 and 111-119, fragments or variants thereof, operably linked to a promoter sequence.

In another aspect, the invention provides a plasmid comprising a polynucleotide selected from the group consisting of any nucleotide sequence of SEQ ID NOs: 95-102 and 111-119, fragments or variants thereof, operably linked to a promoter sequence.

In a further aspect, the invention provides a host cell comprising an isolated polynucleotide sequence, vector or plasmid according to the invention.

Preferably, the host cell is E. coli. E. coli BL21, wherein the polynucleotide further comprises an expression vector pBAD / HisA or λ expression plasmid.

In a further aspect, the present invention comprises (1) culturing the cells of claim 36 under conditions in which a polypeptide comprising FimA , OprF or a fragment or variant thereof is expressed, and (2) recovering the polypeptide. To 110 or 120 to 128 provide a method for producing a recombinant FimA or OprF selected from any of the sequences. The polypeptide may be recovered in soluble or insoluble form.

In another aspect, an isolated polypeptide of the invention is immunologically effective as a vaccine for the prevention or treatment of periodontal disease in a companion animal and comprises the amino acid sequences shown in SEQ ID NOs: 103-110 and 120-128.

In one embodiment, an isolated polypeptide that is immunologically effective as a vaccine for the prevention or treatment of periodontal disease in a companion animal is an amino acid sequence represented by SEQ ID NOs: 103-110 and 120-128 and 95%, 98% or 99% thereof. Homologues having the above sequence identity are included.

In another embodiment, an isolated polypeptide that is immunologically effective as a vaccine for the prevention or treatment of periodontal disease in a companion animal comprises the amino acid sequence represented by SEQ ID NOs: 103-110 and 120-128, or fragments or variants thereof.

In another embodiment, an isolated polypeptide having an amino acid encoded by a DNA molecule that is immunologically effective as a vaccine for the prevention or treatment of periodontal disease in a companion animal is shown in SEQ ID NOs: 95-102 and 111-119. Nucleotide sequences that hybridize under any stringent conditions with any sequence.

In yet further embodiments, an isolated polypeptide comprising at least about 10 consecutive amino acids, immunologically effective as a vaccine for the prevention or treatment of periodontal disease in a companion animal, is any of SEQ ID NOs: 103-110 and 120-128. Fragments of the polypeptide sequence, the polypeptide being alone or in combination with a carrier, is immunologically effective as a vaccine for the prevention or treatment of periodontal disease in an animal. Preferably, the isolated polypeptide comprises at least about 25 amino acids.

Preferably, the isolated polypeptide for preventing or treating periodontal disease in a companion animal is encoded by a DNA molecule comprising a nucleotide sequence comprising fimA (SEQ ID NOS: 95-102 ).

In addition, an isolated polypeptide for preventing or treating periodontal disease in a companion animal is preferably encoded by a DNA molecule comprising a nucleotide sequence comprising oprF (SEQ ID NOs: 111-119 ).

In a preferred embodiment, the isolated polypeptide is a recombinantly expressed polypeptide selected from the group consisting of FimA (SEQ ID NO: 103 to 110) and OprF (SEQ ID NO: 120 to 128).

In another embodiment, the recombinantly expressed polypeptide is fused with a carrier polypeptide. The fusion polypeptide is preferably essentially a poly-histidine or poly-threonine sequence.

In a further aspect, the invention provides a vaccine for the treatment or prevention of periodontal disease in a companion animal comprising an immunologically effective amount of at least one inactivated stained anaerobic bacterium according to the invention and a pharmaceutically acceptable carrier. do.

In another embodiment, the present invention comprises periodontal in a companion animal comprising an immunologically effective amount of at least one inactivated stained anaerobic bacterium according to the invention, at least one other bacterium or virus and a pharmaceutically acceptable carrier. Provided are vaccines for the treatment or prophylaxis of diseases.

In a preferred embodiment, the vaccine comprises an immunologically effective amount of one or more inactivated stained anaerobic bacteria, one or more Canine Distemper Virus (CDV), Canine Adenovirus-2 (CAV-2). ), Canine Parvovirus (CPV), Canine Parainfluenza Virus (CPI) or Canine Coronavirus (CCV), and pharmaceutically acceptable carriers.

In another aspect, the invention provides a vaccine for the treatment or prevention of periodontal disease in a companion animal comprising an immunologically effective amount of at least one polynucleotide molecule according to the invention and a pharmaceutically acceptable carrier.

 In another aspect, the invention provides a vaccine for the treatment or prevention of periodontal disease in a companion animal comprising an immunologically effective amount of at least one polypeptide according to the invention and a pharmaceutically acceptable carrier.

Preferably, the vaccine for the treatment or prevention of periodontal disease in a companion animal comprises an immunologically effective amount of FimA and a pharmaceutically acceptable carrier.

In addition, the vaccine for treating or preventing periodontal disease in a companion animal preferably includes an immunologically effective amount of OprF and a pharmaceutically acceptable carrier.

Bacteria for use in the vaccines of the present invention are Porphyromonas oyster B43, p. Kansul B46, p. Sukhumvitaria B52, p. Whale B69, blood. Sukhumvitaria B97, p. Kanjinjivalis B98, p. Salibosa B104, oh. Dentikanis B106 and p. Endodontalis B114.

In a preferred embodiment, the stained anaerobic bacteria is blood. Whale B43, p. Salibosa B104 and Oh. Dentikanis B106.

In another embodiment, the present invention provides treatment of periodontal disease in a companion animal comprising an immunologically effective amount of at least one inactivated isolated stained anaerobic bacterium according to the invention, a pharmaceutically acceptable carrier and optionally an adjuvant or Provided is a prophylactic vaccine composition.

In another embodiment, the present invention provides a vaccine composition for treating or preventing periodontal disease in a companion animal comprising an immunologically effective amount of at least one polynucleotide molecule according to the invention, a pharmaceutically acceptable carrier and optionally an adjuvant. to provide.

In another further embodiment, the present invention provides a vaccine composition for treating or preventing periodontal disease in a companion animal comprising an immunologically effective amount of at least one polypeptide according to the invention, a pharmaceutically acceptable carrier and optionally an adjuvant. do.

 In another aspect, the present invention provides a method of treating or preventing periodontal disease in a companion animal, comprising administering the vaccine composition according to the present invention to a companion animal in need of treatment or prevention of periodontal disease.

In another aspect, the invention provides a method of diagnosing periodontal disease in a companion animal by analysis of a sample for a bacterium, polypeptide or polynucleotide of the invention, wherein the presence of the bacterium, polypeptide or polynucleotide indicates a disease. Preferably, the analyzing step comprises analyzing the sample using a method selected from the group consisting of PCR, hybridization and antibody detection.

In another aspect, the invention provides treatment of periodontal disease in a companion animal comprising an effective amount of one or more of the inactivated isolated stained anaerobic bacteria, polypeptides or polynucleotides of the invention and a pharmaceutically acceptable carrier in one or more containers. Or a prophylactic composition, the kit further comprising a set of printed instructions indicating that the kit is useful for treating or preventing periodontal disease in a companion animal. The kit may further comprise means for dispensing the composition.

In another aspect, the present invention provides SEQ ID NOs: 86-94, 95-102, which hybridize under high stringency conditions with the complement of any of the nucleotide sequences shown in SEQ ID NOs: 86-94, 95-102 and 111-119 in one or more containers. And an isolated DNA molecule comprising a nucleotide sequence of at least about 15 contiguous nucleotides selected from any one of 111-119, and any nucleotide shown in SEQ ID NOs: 86-94, 95-102, and 111-119 in a second container. An isolated nucleotide sequence comprising at least about 15 contiguous nucleotides selected from the complement of any nucleotide sequence shown in SEQ ID NOs: 86-94, 95-102, and 111-119, which hybridizes under high stringency conditions. A set of printed molecules containing DNA molecules, indicating that the kit is useful for detecting porphyromonas species It provides a kit further includes a manual. Such methods can generally be used in all mammals, including humans.

In another aspect, the invention provides a protein, and kit having an amino acid sequence comprising at least 30 contiguous amino acids, wherein the polypeptide in one or more containers is encoded by any of the nucleotide sequences of SEQ ID NOs: 95-102 and 111-119. Kits are provided that include a statement indicating utility in the detection of Porphyromonas spp. The kit may further comprise a second polypeptide that is an antibody conjugated to an enzyme that catalyzes the color difference system. The enzyme is preferably selected from the group consisting of alkaline phosphatase and horseradish peroxidase. The kit may further comprise a reagent for color difference or chemifluorescence analysis.

In a further aspect, the invention provides an isolated DNA comprising a nucleotide sequence of at least about 15 consecutive nucleotides selected from any one of SEQ ID NOs: 86-94, 95-102 and 111-119, or complements thereof, in one or more containers. The hybridization kit further comprises a molecule, and a set of instructions indicating that the kit is useful for the detection of Porphyromonas species, wherein hybridization is specific for Porphyromonas species. Preferably, hybridization is performed under high stringency conditions.

In another further aspect, the present invention provides a method for assessing the efficacy of a vaccine against one or more periodontal pathogenic bacteria in an animal, particularly a dog.

In another further aspect, the invention provides a method for determining alveolar bone, in which the clinical signs measured in an animal are quantified through increased levels of one or more periodontal pathogenic bacteria in gingival fluid, plaque, infected bone or gingival sulcus, or radiometric Provided are methods for assessing the efficacy of a vaccine against one or more periodontal pathogenic bacteria, including a change in amount.

A strain of any of the bacteria, polynucleotides, polypeptides, vaccines, vaccine compositions or kits of the invention designated ATCC 57100. Go ahead. See, et al., Fournier, D. et al., “ Porphyromonas gulae sp. nov., an Anaerobic, Gram-negative, Coccibacillus from the Gingival Sulcus of Various Animal Hosts ", International Journal of a Systematic and Evolutionary Microbiology (2001), 51, 1179-1189, Hirasawa and Takada," Porphyromonas gingivicanis sp . nov. and Porphyromonas crevioricanis sp. nov., Isolated from Beagles ", International Journal of Systemic Bacteriology, pp. 637-640, (1994)], U.S. Patent 5,710,039 or 5,563,063, or International Application PCT / AU98 with Publication No. WO 99/29870. It does not include any bacteria, polynucleotides or peptides described in / 01023.

1 is a neighbor-joining phylogenetic tree for a representative Bacteroidetes class. The phylogenetic branching was generated using the CLUSTAL X version 1.81 and the NJ Plot software program (both available at ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/). The branching map is based on the Escherichia coli 16S rRNA gene sequence (accession number J01695; data not shown). Bootstrap analysis was performed with 1000 iterations. Bootstrap values are presented graphically (●>950;■>850;○>700;□>500; no indication <500). Scale bars represent 0.01 substitutions per nucleotide position. The arrow is oh. Indicates the position of Dentikanis B106 ^T. Access number: p. Zhiblis ATCC 33277, J01695; blood. Whale B243, AF285874; blood. Kansul VPB 4875, X76260; blood. Salibosa NCTC 11632, L26103; blood. Endodontalis ATCC 35406, AY253728; tea. T. forsythensis ATCC 43037, AB035460; Bacteroides cf. Forsythus oral clone BU45, AF385565; ratio. B. merdae ATCC 43184T, X83954; ratio. B. distasonis ATCC 8503, M86695; Horse dung bacteria 118ds10, AY212569; D. D. shahii strain CCUG 43457, AJ319867; Uncultured Bacteroidaceae clones: Rs-P82, AB088919; Uncultivated bacteria cadhufec 059h7, AF530302; ratio. B. splanchnicus NCTC 10825, L16496; Five. Denticanis B106 ^T , AY560020; Bacteroidetes spp. Oral clone FX069, AY134906; Uncultured bacteria SHA-38, AJ249105; a. Putredinis ATCC 29800, L16497; egg. R. microfusus ATCC 29728, L16498; ratio. B. denticanium B78, AY549431; ratio. P. fragilis ATCC 25285T, X83935; ratio. B. thetaiotaomicron Strain 17.4, AY319392; ratio. B. acidofaciens strain A37, AB021163; blood. P. bivia ATCC 29303. L16475; blood. Nigrescens ATCC 25261, L16479; blood. P. intermedia ATCC 25611, L16468; blood. P. denticola ATCC 35308, L16467; And blood. P. buccae ATCC 33690, L16478.

FIG. 2 is a neighborhood- linked phylogenetic map of clinical isolates of Odoribacter denticanis . The phylogenetic branching was generated as in FIG. 1. The branching map is based on Porphyromonas jinjivalis ATCC 53977 16S rRNA gene sequence (accession number L16492; data not shown). Size bars represent 0.01 substitutions per nucleotide position. Access number: Oh. Denticanis B106 ^T , AY560020; Five. Denticanis B113, AY560022; Five. Denticanis B150, AY560027; Five. Denticanis B155, AY560030; Five. Denticanis B172, AY560033; Five. Denticanis B183, AY560035; Bacteroidetes spp. Oral clone FX069, AY134906; Uncultured bacteria Kaffupek 059h7, AF530302; ratio. Splancinicus NCTC 10825; And uncultured bacteridaidae clones: Rs-P82, AB088919.

FIG. 3 is a neighborhood-linked phylogenetic branching of a representative Porphyromonas gingivalis FimA protein family (Class I to Class V). The phylogenetic branching was generated using CLUSTAL X version 1.81 and NJ plot software program. The branching is based on the Escherichia coli CFT073 fimbrillin protein (accession NP_757241; data not shown). Bootstrap analysis was performed with 1000 iterations. Bootstrap values are presented graphically (●>950;■>850;○>700;□>500; no indication <500). Size bars indicate 0.05 substitutions per amino acid position. FimA access number: Oh. Denticanis B106 ^T , AY573801; blood. Seriously ballis HG1691, Q93R80; blood. Zhiblis BH18 / 10, JN0915; blood. Jinjivalis ATCC 33277, P13793; blood. Zhiblis OMZ314, BAA04624; blood. Earnest Ballis OMZ409, Q51822; blood. Zhiblis 6/26, Q51826; blood. Seriously Ballis ATCC 49417, Q51825; blood. Seriously ballis W83, AAQ67087; blood. Seriously ballis HG564, Q51827; And blood. Jinji Balis HNA-99, Q9S0W8.

4 (A) p. Dolphin scanning electron micrograph of B43 and (B) o. Scanning electron micrograph of Dentikanis B106 ^T. Size bars indicate distances of 1000 nm.

FIG. 5 is a graph showing the results of growth studies identifying “animal product-free” media sustaining growth of Porphyromonas oyster B43. The following media were tested: ME-complete, ME-hemin, ME-vitamin K, both ME-hemin and vitamin K, PYG-complete, PYG-hemin, PYG-vitamin K, PYG-hemin and vitamin K And BHI.

FIG. 6 is a graph showing average bone loss in mice generated by super infection with the indicated bacterial species. Mice were challenged with sham challenge or labeled bacteria. Net bone loss indicates bone loss above and below the value observed in the Siamese challenge group. Dashed line represents net bone loss in the positive control. Standard error is indicated.

FIG. 7 is a graph showing percent bone loss in mice generated by super infection with the indicated bacterial species. Mice were challenged with Siamese or labeled bacteria. % Bone loss is based on net bone loss from the positive control set as 100% bone loss.

8A and 8B show E. coli using anti-Xpress ™ epitope serum (Invitrogen). Recombinant blood expressed from pBAD-HisA in E. coli BL21. As for B43 FimA, Figure 8A shows the SDS PAGE analysis and Figure 8B shows the Western blotting analysis. Abbreviation: Std. in kDa, standard; Ind, derived; Sol, soluble fraction; And Insol, insoluble fraction. The arrow head indicates the location of rFimA.

9 is E. Recombinant blood expressed from expression plasmids in E. coli BL21 cells. The photograph shows the SDS-PAGE analysis of the B43 oprF. T represents the time after raising the culture from 30 ° C to 42 ° C. Standards of molecular mass size are given in kDa. The arrow head points to the location of rOprF.

10 is a graph showing the results of a homologous vaccine efficacy study based on net bone loss. Homeopathic vaccine efficacy studies are based on net bone loss. Mice were vaccinated using the following vaccine antigens and RIBI MPL + TDM adjuvant (except group C. Groups used Freunds complete and incomplete): A, B and E, PBS alone; C and D, formalin-activated blood. Seriously ballis 53977; F, formalin-activated blood. Whale B43; G, BEI-activated blood. Whale B43; H, heat-inactivated blood. Whale B43; And I, aeration-activated blood. Whale B43. Mice are challenged with Siamese (Group A) or blood in 1% carboxymethylcellulose. Zhiblis 53977 (hatched boxes) or blood. Whales were challenged with B43 (black boxes). Standard error measurements are indicated.

11 is blood based on percent bone loss. This is a graph showing the efficacy of the Genjivalis 53977 homologous vaccine. Mice were vaccinated with the following vaccine antigens and RIBI MPL + TDM adjuvant (except group C. Freund's complete and incomplete was used in group C): A and B, PBS alone; C and D, formalin-activated blood. Genjivalis 53977. Mice were challenged with A in PBS, or B-D in blood in 1% carboxymethylcellulose. Infection was with Genjivalis 53977.

12 is blood based on percent bone loss. This is a graph showing the study of efficacy of the B43 homologous vaccine. Mice were vaccinated using the following vaccine antigens and RIBI MPL + TDM adjuvant: A and B, PBS alone; C, formalin-activated blood. Whale B43; D, BEI-activated blood. Whale B43; E, heat-inactivated blood. Whale B43; And F, aeration-inactivated blood. Whale B43. Mice were challenged with A in PBS, or B-F in 1% carboxymethylcellulose. Gulp was challenged with B43.

13 is a graph showing the results of a heterologous vaccine efficacy study based on net bone loss. Mice were vaccinated using the following vaccine antigens and RIBI MPL + TDM adjuvant: A to E, PBS alone; F to I, formalin-activated blood. Whale B43; J, formalin-activated blood. Salibosa B104 and formalin-activated o. Denticanis B106. Mice are challenged with Siamese (A) with PBS, or blood in 1% carboxymethylcellulose. Whale B43 (B and F; box painted in black), blood. Whale B69 (C, G and J; boxes hatched with diagonal lines), blood. Salibosa B104 (D and H; sideways boxed) or o. Infected with Dentikanis B106 (E and I; boxes with polka dots). Standard error measurements are indicated.

14 is blood based on percent bone loss. This is a graph showing the results of a heterologous vaccine efficacy study in the Bula B43 challenge group. Mice were vaccinated using the following vaccine antigens and RIBI MPL + TDM adjuvant: A and B, PBS alone; C, formalin-activated blood. Whale B43. Mice were challenged with A in PBS, or B and C in blood in 1% carboxymethylcellulose. Gulp was challenged with B43.

Figure 15 is blood based on percent bone loss. This is a graph showing the results of a heterologous vaccine efficacy study in the Bulwa B69 challenge group. Mice were vaccinated using the following vaccine antigens and RIBI MPL + TDM adjuvant: A and B, PBS alone; C, formalin-activated blood. Whale B43; Or D, formalin-activated blood. Salibosa B104 and formalin-activated o. Denticanis B106. Mice were challenged with A in PBS, or B to D in blood in 1% carboxymethylcellulose. Boil was challenged with B69.

16 is blood based on percent bone loss. A graph showing the results of a heterologous vaccine efficacy study of Salibosa B104 challenge group. Mice were vaccinated using the following vaccine antigens and RIBI MPL + TDM adjuvant: A and B, PBS alone; Or C, formalin-inactivated blood. Whale B43. Mice were challenged with A in PBS, or B and C in blood in 1% carboxymethylcellulose. Infected with Salibosa B104.

FIG. 17 is a blot based on percent bone loss. This is a graph showing the results of a heterologous vaccine efficacy study of Dentikanis B106 challenge group. Heterologous vaccine efficacy studies Based on percent bone loss for Dentikanis B106 infected group. Mice were vaccinated using the following vaccine antigens and RIBI MPL + TDM adjuvant: A and B, PBS alone; Or C, formalin-activated blood. Whale B43. Mice were challenged with A in PBS or B and C in O. carboxymethylcellulose. It was challenged with Dentikanis B106.

18 shows recombinant blood. A graph showing serological results using FimA-specific ELISA in mice vaccinated with native B43 FimA or saline. Each of the 16 mice was vaccinated with saline (A) or rFimA / QuilA / cholesterol (B). Pooled sera were tested for FimA-specific antibodies via FimA-specific ELISA. The minimum dilution tested was 1: 100. A titer of 50 was given to any sample that showed a negative result at this dilution rate.

19 shows recombinant blood. A graph showing serological results using OprF-specific ELISA in mice vaccinated with native B43 OprF or saline. Each of the 16 mice was vaccinated with saline (A) or rOprF / QuilA / cholesterol (B). Pooled sera were tested for OprF-specific antibodies via OprF-specific ELISA. The minimum dilution tested was 1: 100. A titer of 50 was given to any sample that showed a negative result at this dilution rate.

20 is a graph showing bone loss at weeks 0, 6, and 12 after challenge. T01 group is represented by dogs 3559424, 3592669, 3672859, 3673375 and 3691926, and T02 group is represented by dogs 3389600, 3628884, 3653552, 3657396, 3690164.

FIG. 21 shows the T01 group (vaccinated and challenged, or Vx / Ch), T02 group (Shammer vaccinated) at weeks 0, 3, 6, 9 and 12 after challenge Graph showing bone reactivity scores for challenged, or non-Vx / Ch) and T03 (Siamese vaccinated and Siamese challenged, or non-Vx / non-Ch). The statistical significance of the treatment effect is also indicated.

22 shows T01 (Vx / Ch), T02 (non-Vx / Ch) and T03 (non-Vx / non-Ch) at weeks 0, 3, 6 and 9 after challenge. Graph showing bone responsiveness score for). Statistical significance between the T01 and T02 groups was indicated.

Figures 23A-23D show from one dog at Week 0 (23A), Week 3 (23B), Week 6 (23C) and Week 9 (23D) after challenge in group T01 (Vx / Ch). Radiographic image. 23E-23H show 1 dog at week 0 (23E), week 3 (23F), week 6 (23G) and week 9 (23H) after challenge in group T02 (non-Vx / Ch). From radiographic images.

24A-24C are graphs showing average systemic responses to T01 (Vx / Ch) and T02 (non-Vx / Ch) groups. The score is based on the evaluation result of grading the physical activity level of all dogs in each group. 24D-24F are graphs showing mean local responses to T01 group (Vx / Ch) and T02 group (non-Vx / Ch). The score is based on an evaluation of grading swelling levels present in all dog injection sites in each group.

Bacterial isolate

The present invention provides isolated anaerobic bacteria that cause periodontal disease and various other diseases and clinical symptoms in companion animals and are identified by 16S rRNA DNA sequences. More specifically, the bacterium is selected from the genus Porphyromonas.

Preferably, the isolated bacteria of the present invention are blood. Whale B43, p. Kansul B46, p. Sukhumvitaria B52, p. Whale B69, blood. Sukhumvitaria B97, p. Kanjinjivalis B98, p. Salibosa B104, oh. Dentikanis B106 and p. Endodontalis B114, and other species or strains are included in the present invention. In a preferred embodiment, the isolated bacteria of the invention can be identified by their 16S rRNA DNA sequences shown in SEQ ID NOs: 86-94.

Diseases caused by infection with the bacteria of the present invention include, but are not limited to, companion animal periodontal disease, companion animal oral odor (bad breath), bovine foot rot, dog coronary heart disease, and systemic infection of the dog. . Bacteria in Porphyromonas also include coronary heart disease, mumps, oral odor, gingivitis, periodontitis, stroke, atherosclerosis, hyperlipidemia, bacterial vaginosis, delayed intrauterine development (IUGR), and increased incidence of premature birth of low birth weight infants. It is related to the disease.

The present invention provides isolated polynucleotides and isolated polypeptide molecules of Porphyromonas spp. More particularly, the present invention provides an isolated polynucleotide molecule having the nucleotide sequence of the porphyromonas species fimA and oprF genes or their degenerate variants and the amino acid sequence of each of the FimA and OprF proteins encoded by such genes. An isolated polypeptide molecule is provided.

In addition, the present invention provides at least about 90% homology, preferably at least about 95%, most to any of SEQ ID NOs: 95 to 102 and SEQ ID NOs: 111 to 119 as determined using any known standard identity algorithm. Preferably a polynucleotide sequence having at least 99% sequence identity is provided. The present invention also provides a polynucleotide sequence that hybridizes under stringent conditions to the complement of any of the polynucleotide sequences set forth in SEQ ID NOs: 95-102 and 111-119.

In another specific embodiment, nucleic acids are provided that are hybridizable under high stringency conditions to any of the polynucleotide sequences shown in SEQ ID NOs: 86-102 and SEQ ID NOs: 111-119, or complements thereof. As a non-limiting example, the procedure using these high stringency conditions for more than 90 nucleotide hybridization regions is as follows: prehybridization of a filter containing DNA was performed using 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 Perform 8 hours to overnight at 65 ° C. in a buffer consisting of mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 500 μg / mL denatured salmon sperm DNA. The filter is hybridized at 65 ° C. for 48 hours in a prehybridization mixture containing 100 μg / mL denatured salmon sperm DNA and 5 × 10 ⁶ to 20 × 10 ⁶ cpm of ³² P-labeled probes. The filter wash is performed for 1 hour at 37 ° C. in a solution containing 2 × SSC, 0.01% PVP, 0.01% Picol and 0.01% BSA. Subsequently, self-radiography is performed after washing in 0.1X SSC for 45 minutes at 50 캜.

Other stringent conditions that can be used depend on the nature of the nucleic acid (eg length, GC content, etc.) and the purpose of hybridization (detection, amplification, etc.) and are known in the art. For example, stringent hybridization of approximately 15 to 40 bases of oligonucleotides to complementary sequences in a polymerase chain reaction (PCR) is performed under the following conditions: salt concentration of 50 mM KCl, 10 mM Tris-HCl Buffer concentration, Mg ²⁺ concentration of 1.5 mM, pH of 7-7.5 and annealing temperature of 55 ° C. to 60 ° C.

In a preferred specific embodiment, the washing conditions after hybridization are as follows: Each membrane was washed twice at 30 ° C. in 45 mM 40 mM sodium phosphate, pH 7.2, 5% SDS, 1 mM EDTA, 0.5% bovine serum albumin. Each is then washed four times in sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA for 30 minutes. For high stringency hybridization, the membranes were further washed four times for 30 min in 40 mM sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA at 55 ° C., respectively, then each at 65 ° C., sodium phosphate, pH 7.2, 1%. SDS, wash 4 times for 30 min in 1 mM EDTA.

The present invention further provides vaccines and vaccine formulations useful for the treatment or prevention (ie, tolerating resistance) of periodontal disease in companion animals when administered to a companion animal in therapeutically effective amounts.

In one embodiment, the present invention provides a vaccine comprising one or more attenuated whole cell porphyromonas species preparations (modified live vaccine) or a vaccine comprising inactivated whole cell porphyromonas species preparations (bacterin) do. In a preferred embodiment, the invention provides inactivated whole cell preparations of three or more porphyromonas species, for example blood. Whale B43 and blood. Salivasa B104 and Oh. A vaccine containing a combination of dentikanis B106 is provided. Bacterial cells can be inactivated using a variety of agents such as formalin, binary ethylenimine (BEI) or beta-propriolactone. Preferably, formalin is used as the deactivator.

In another embodiment, the vaccine comprises a subunit fraction of Porphyromonas species capable of inducing an immune response.

In a preferred embodiment, the vaccine of the invention comprises one or more subunit polypeptides or fragments or variants thereof, or one or more isolated polynucleotide sequences or fragments or variants thereof.

Attenuated vaccine (modified live vaccine) or inactivated vaccine (bacterin) or isolated subunit polypeptide or isolated polynucleotide may be present with other known vaccine formulation components, eg, compatible adjuvants, diluents or carriers. have.

Definition and abbreviation

The term "ORF" refers to the "open reading frame" of a gene, ie the coding region.

The term "% sequence identity" for nucleotide sequences and polypeptide sequences is determined by comparing two sequences that are optimally aligned over a comparison window, where the optimal alignment provides the highest ranked match, and the nucleotide or amino acid Adducts may be introduced into the test sequence or reference sequence. The percent identity is determined by calculating the proportion of identical nucleotides between the test sequence and the reference sequence at each position over the entire sequence. Optimal sequence alignment and percent identity can be determined manually or more preferably by computer algorithms including but not limited to TBLASTN, BLASTP, FASTA, TFASTA, GAP, BESTFIT and CLUSTALW, and the like ([ Altschul et al., 1990, J. MoI. Biol. 215 (3): 403-10, Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-8, Thompson, et al., 1994, Nucleic Acids Res. 22 (22): 4673-80, Devereux et al., 1984, Nuc.Acids.Res. 12: 387-395, Higgins, et al., 1996, Methods Enzymol. 266: 383-402. Preferably, the NCBI Blast server (http://www.ncbi.nlm.nih.gov) set with default parameters is used to search multiple databases for homologous sequences.

As used herein, the term “heterologous” means derived from bacteria of different species or different strains.

As used herein, the terms “homology,” “homologous,” and the like refer to the degree of identity shared between polynucleotide sequences or between polypeptide sequences.

The term "homologous" as used referring to a bacterial species means a bacterium of the same species or of the same strain.

As used herein, the term “host cell” refers to a bacterial or eukaryotic cell that carries a plasmid, virus or other vector.

As used herein, the term “isolated” means isolated alone or from its natural environment in a heterogeneous host cell or chromosome or vector (eg plasmid, phage, etc.).

The terms “isolated anaerobic bacteria”, “isolated bacteria”, “isolated bacterial strains” and the like refer to compositions that are substantially free of microorganisms other than bacteria, for example in culture, such as when the bacteria are isolated from their natural environment. Refers to.

The term "isolated polynucleotide" refers to a composition in which the isolated nucleotide comprises at least 50% by weight of the composition. More preferably, the isolated polynucleotide comprises about 95%, most preferably 99% by weight of the composition.

The term "isolated polypeptide" refers to a composition in which the isolated polypeptide comprises at least 50% by weight of the composition. More preferably, the isolated polypeptide comprises about 95%, most preferably 99% by weight of the composition.

As used herein, the term “functionally equivalent” refers to a recombinant polypeptide that can be recognized by an antibody specific for a natural polypeptide produced by a bacterium that causes periodontal disease in a companion animal, or a natural protein from an endogenous bacterium. Refers to a recombinant polypeptide capable of causing or eliciting an immunological response substantially similar to an immunological response. Thus, antibodies produced against functionally equivalent polypeptides also recognize natural polypeptides produced by bacteria that cause periodontal disease in companion animals.

The term “immunogenic” refers to the ability of a protein or polypeptide to elicit an specifically directed immune response against bacteria that cause periodontal disease in a companion animal.

The term “antigenicity” refers to the ability of the protein or polypeptide to be immunospecifically bound by an antibody produced against a particular protein or polypeptide.

As used herein, the term “antibody” refers to an immunoglobulin molecule capable of binding to an antigen. The antibody may be a polyclonal mixture or may be monoclonal. The antibody may be an intact immunoglobulin derived from a natural source or recombinant source, or may be an immunoreactive portion of an intact immunoglobulin. Antibodies may exist in various forms, including, for example, Fv, Fab ', F (ab') _2, and the like, and may also exist in single chain form.

As used herein, the term “companion animal” refers to any non-human animal that has been raised as a pet. These include, but are not limited to, dogs, cats, horses, sheep, rabbits, monkeys, and rodents, including mice, rats, hamsters, gerbils and ferrets.

As used herein in connection with vaccines, the terms “protecting”, “protecting” and the like refer to preventing or reducing the symptoms of a disease caused by an organism from which the antigen (s) used in such vaccines are derived. it means. The terms "protection", "protecting" and the like also mean that the vaccine can be used to "treat" a disease already occurring in a subject or one or more symptoms of the disease.

The term “therapeutically effective amount” refers to an amount sufficient for a bacterium or subunits thereof (eg, polypeptides, polynucleotide sequences) and combinations thereof to cause an immune response in a subject to be administered. Immune responses may include, but are not limited to, cellular and / or humoral immune induction.

The term “infectious disease prevention” refers to preventing or inhibiting the replication of bacteria that cause periodontal disease in a companion animal, inhibiting the transmission of such bacteria, or preventing the bacteria themselves from establishing in their host, or caused by an infection. To alleviate the symptoms of the disease. If the bacterial load is reduced, the treatment is considered therapeutic.

The term "pharmaceutically acceptable carrier" refers to a carrier medium that does not interfere with the biological activity efficiency of the active ingredient and is not toxic to the subject to be administered.

The term “therapeutic agent” refers to any molecule, compound or therapeutic agent, preferably an antimicrobial molecule or antimicrobial compound, which aids in the treatment of a bacterial infection or disease or condition caused thereby.

The term “fragment or variant thereof” refers to a partial nucleotide sequence or amino acid sequence according to the invention. Fragments or variants of the polypeptides provided herein are preferably capable of eliciting humoral and / or cellular immune responses in companion animals. The term “fragment or variant thereof” includes analogues. The term “fragment or variant thereof” includes mutant polynucleotides that may carry one or more mutations that are deletions, insertions, or substitutions of nucleotide residues. The term “fragment or variant thereof” includes allelic variants.

Isolation and Characterization of Porphyromonas Species

The bacteria provided by the present invention can be obtained using known sampling, culturing and isolation techniques. For example, microbial samples can be obtained from companion animal populations that exhibit periodontal disease, such as dogs and cats. Evidence of periodontal disease can be observed using known measurements, for example dogs with periodontal pockets> 3 mm and cats with periodontal pockets> 2 mm. Parameters known to characterize periodontal disease, such as tooth index (gingiva index and periodontal index) and periodontal pocket depth, can be measured against a sample population of animals. Individual samples can be obtained from the periodontal pockets of certain animals, which can be maintained under anaerobic conditions and cultured using various known culture media.

By characterizing clinical isolates using known techniques such as numerous biochemical tests and 16S rRNA DNA sequencing, their genus and species can be determined. Individual isolates can be transferred to plates and antibiotic discs (Anaerobe Systems) placed on the agar surface to determine the antibiotic resistance pattern of each isolate. Purified colonies may be subjected to known indole and catalase tests (Unaero Systems). Lipase and resitinase production patterns for individual isolates can be determined.

The types of isolates can be classified based on their 16S rRNA DNA sequences. Individual well isolated colonies can be utilized as templates for polymerase chain reaction (PCR) amplification of 16S rRNA regions using, for example, primers D0056 and D0057 (SEQ ID NO: 1 and SEQ ID NO: 2, Table 1). The resulting PCR product can be pooled by purification and isolation using a commercially available PCR preparation kit (Promega Corp .; Madison, Wisconsin). The purified PCR product can then be desalted and subjected to DNA sequencing. The generated DNA sequences can be used to search for available DNA databases. Thereafter, the type of bacterial isolate can be classified based on the closest match result identified by the database search.

Note : The nucleotides in the subcase are not present in the target DNA sequence. These are added to the 5 'region of the primer to aid cloning. NA, not applicable

doing Pet periodontal isolates were deposited on 9 August 2001 at the American Type Culture Collection (ATCC), Boulevard 10801, Manassas University, Virginia, USA 0. p. Oyster B43 (PTA-3618), p. Kansul B46 (PTA-3619), p . Circumdentaria B52 (PTA-3620), p. Oyster B69 (PTA-3621), p. Circumdentaria B97 (PTA-3622), p. Kanjinji Balis B98 (PTA-3623), p. Salibosa B104 (PTA-3624), oh. Dentikanis B106 (PTA-3625) and p. Endodontalis B114 (PTA-3626). In a preferred embodiment of the invention, the isolated polynucleotide molecules of the invention have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 86-102 and 111-119. Preferred polypeptides of the invention have an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-110 and 120-128.

Porphyromonas  Nucleotide sequence Cloning

There are several known methods or techniques that can be used to clone the porphyromonas nucleotide sequences of the present invention. For example, the sequence can be isolated into fragments by restriction enzymes and cloned into cloning and / or expression vectors, amplified by PCR and cloned into cloning and / or expression vectors, or a combination of both methods. Can be cloned.

Standard molecular biology techniques known in the art and not specifically described herein are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), Watson et al., Recombinant DNA, Scientific American Books, New York, Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998), and US Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057. Polymerase chain reaction (PCR) can generally be performed as described in PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, CA (1990).

Examples of methods useful for the cloning and sequencing of polynucleotides of the invention are provided in the Examples below.

fimA And oprF Coded Polypeptides and Proteins

The present invention is directed to prokaryotic and eukaryotic expression systems, including vectors and host cells, which can be used to express both truncated and full-length (natural protein) forms of recombinant polypeptides expressed by the nucleotide sequences of the invention. Includes use.

In a preferred embodiment of the invention, the isolated polynucleotide molecules of the invention are selected from SEQ ID NOs: 95 to 102 and 111 to 119, or synonymous variant sequences thereof, and the amino acids of SEQ ID NOs: 103 to 110 and 120 to 128, respectively Has a nucleotide sequence encoding the corresponding polypeptide selected from the sequence .

Various host-expression vector systems can be used to express polypeptides of the invention. In addition, such host-expression systems represent vehicles in which the coding sequence can be cloned and subsequently purified. The invention also provides host cells capable of expressing the encoded polypeptide gene products of the invention when transformed or transfected with appropriate vectors or nucleotide sequences. Such host cells include microorganisms such as bacteria (eg, E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences; Yeast transformed with recombinant yeast expression vectors containing gene product coding sequences (eg, Saccharomyces, Pichia); Insect cell systems infected with recombinant virus expression vectors (eg, baculovirus) containing coding sequences; Recombinant plasmid expression vector containing or coding with a recombinant viral expression vector containing a coding sequence (e.g., cauliflower mosaic virus (CAMV); tobacco mosaic virus (TMV)) or containing a coding sequence Plant cell systems transformed with (eg, Ti plasmids); Or recombinant containing a promoter derived from the genome of a mammalian cell (eg metallothionein promoter) or a promoter derived from a mammalian virus (eg adenovirus late promoter, vaccinia virus 7.5K promoter). Mammalian cell systems (eg, COS, CHO, BHK, 293, 3T3) that carry expression constructs include, but are not limited to.

In a preferred embodiment, the expression system is a bacterial system. Many expression vectors can be advantageously selected depending on the desired use of the product to be expressed. For example, where a large amount of polypeptide is to be produced for vaccine composition production and antibody development, a vector may be preferred, for example, which expresses high levels of readily purified fusion protein products. Preferably, the vector contains a promoter that directs the expression of the inducible gene. Suitable vectors include E. coli, wherein the coding sequence can be fused in-frame to a sequence encoding a plurality of (eg , six) histidine residues. Coli pET expression vectors (Studier and Moffatt, 1986, J. Mol. Biol. 189: 113, Rosenberg et al., 1987, Gene 56: 125-135); Novagen, Madison, Wisconsin ); PBAD vectors in which heterologous proteins can be expressed under the control of arabinose inducible proteins (Guzman et al., 1995, J. Bact. 177: 4121-4130); And pGEX vectors (Pharmacia Biotech, USA) used to express heterologous polypeptides as fusion proteins using glutathione S-transferase (GST). The fimA or oprF sequences of the invention can be cloned into λ expression vectors and expressed in λ ⁻ bacterial strains. In a preferred method, the bacterial strain is E. coli. E. coli BL21 (Gibco-BRL, USA). Preferably, as a vector that can be used, pLEX expression vectors (LaVallie et al., 1992, Bio / Technology 11: 187-193), Meischendahl et al., 1986, Bio / Technology 4: 802-808; Invitrogen) and pRIT2T expression vectors (Nilsson et al., 1985, EMBO 4: 1075, Zabeau and Stanley, 1982, EMBO 1: 1217; Pharmacia Biotech), but are not limited thereto. Other vectors and bacterial strains can be used and they are known to those skilled in the art.

Antibody production

The antibody may be monoclonal, polyclonal or recombinant antibody. Typically, an antibody is prepared for an immunogen or portion thereof, eg, a synthetic peptide recombinantly prepared by a sequenced synthetic peptide or cloning technique, or a natural gene product and / or portion thereof that can be isolated and used as an immunogen. Can be. Immunogens are generally described in Harlow and Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antidbody Engineering-A Practical Guide, WH Freeman and Co., 1992. As described, it can be used to produce antibodies by standard antibody production techniques known to those skilled in the art. Antibody fragments may also be prepared from antibodies by methods known to those of skill in the art and include Fab, F (ab ') ₂ and Fv.

In antibody production, screening for the desired antibody can be performed by standard methods known in the art of immunology. Techniques not specifically described are generally described in Sites et al. (Eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, WH Freeman and Co., New York (1980). In general, ELISA and western blotting are the preferred immunoassay types. Both assays are known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used for these assays. Antibodies may be bound to a solid support substrate, conjugated with a detectable moiety, or bound and conjugated as is known in the art (see the general discussion on the conjugation of fluorescence or enzyme residues, see Johnstone & Thorpe). , Immunochemistry in Practice, Blackwell Scientific Publications, Oxford, 1982). Binding of antibodies to solid support substrates is also known in the art (see the general discussion of Harlow & Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York, 1988) and Borrebaeck, Antibody Engineering- A Practical Guide, WH Freeman and Co., 1992). Detectable residues contemplated for use in the present invention include fluorescent, metal, enzyme and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein, rhoda Min, tritium, ¹⁴ C and iodine, but are not limited thereto.

If appropriate, other immunoassays such as radioimmunoassay (RIA) can be used as known in the art. Useful immunoassays are extensively described in patents and scientific publications (eg, US Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 1st). 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771 and 5,281,521, and Sambrook et al, Molecular Cloning: A Laboratory Manual Springs Harbor, New York, 1989).

Detection, diagnosis and prevention Kit

The invention also provides a kit for detecting Porphyromonas spp. This kit comprises a porphyromonas organism, polypeptide or porphyromonas of the invention. Reagents used to analyze a sample for the presence of a nucleotide sequence, wherein the nucleotide The presence of the sequence indicates that the organism is present. This method is useful because the disease can be diagnosed before the symptoms appear, thus preventing the onset of the disease before the patient is harmed. Porphyromonas species The presence of bacteria, polypeptides or nucleotide sequences can be determined using antibodies, PCR, hybridization and other detection methods known to those skilled in the art.

In one embodiment, the kit provides a reagent for detecting an antibody against Porphyromonas. In certain embodiments, the kit may comprise a set of instructions or labels indicating that the kit is useful for the detection of Porphyromonas spp. At a minimum, the kit comprises a protein having an amino acid sequence (including at least 30 contiguous amino acids) of any of the polypeptides of SEQ ID NOs: 103-110 and 120-128 in one or more containers. In one embodiment, the kit also includes a secondary antibody. In a preferred embodiment, the secondary antibody is conjugated to a detectable moiety, for example an enzyme catalyzing a colorimetric or chemiluminescent reaction such as alkaline phosphatase or horseradish peroxidase. In further embodiments, the kit comprises a reagent used to perform colorimetric or chemiluminescence assays.

In other embodiments, the kit is porphyromonas Provided are reagents for detection of nucleic acids. In one embodiment, the kit is porphyromonas A reagent for use in PCR detection of nucleic acids is provided, wherein the sequence of any one of the polypeptides of SEQ ID NOs: 103 to 110 or 120 to 128 (5, 10, 15, 20, 25, or 30 or more in one or more containers under high stringent conditions) Amino acid sequence) or a first isolated DNA molecule comprising a fragment having at least about 15, 20, 25, or 30 nucleotides that hybridizes to a DNA molecule encoding a polypeptide comprising a complete amino acid sequence; And encoding a polypeptide comprising a sequence of any one of the polypeptides of SEQ ID NOs: 103 to 110 or 120 to 128 (at least 5, 10, 15, 20, 25, or 30 contiguous amino acid sequences) or a complete amino acid sequence under high stringency conditions; A second isolated DNA molecule comprising a fragment having at least 15, 20, 25, or 30 nucleotides, hybridized to a DNA molecule complementary to the DNA molecule, wherein the first and second DNA molecules comprise SEQ ID NOs: 1-9 It can be used to specifically amplify Porphyromonas species nucleic acid encoding 16S rRNA encoded by a DNA molecule selected from the group consisting of:

In a further embodiment, the invention SEQ ID NOS: 86-94, 95 hybridizes in one or more containers to the complement of any one of the nucleotide sequences shown in SEQ ID NOs: 86-94, 95-102 and 111-119 under strict conditions To isolated DNA molecules comprising a nucleotide sequence selected from any one of from 102 to 111 and from 119 to 119, including at least about 15 contiguous nucleotides, and under high stringent conditions, SEQ ID NOs: 86 to 94, 95 to 102 and 111 to A nucleotide sequence (including at least about 15 contiguous nucleotides) selected from the complement of any one of the nucleotide sequences shown in SEQ ID NOs: 86-94, 95-102 and 111-119, which hybridizes to any one of the nucleotide sequences represented by 119; To provide a kit comprising a second isolated DNA molecule in a second container which is an isolated DNA molecule, the kit A documentation set that represents the useful for the detection of Pseudomonas species formate fatigue further includes.

Vaccine Formulation and Methods of Administration

By administering an effective amount of the vaccine of the present invention to a companion animal, it is possible to treat the periodontal disease in a companion animal therapeutically, to impart or to prevent it. The vaccine of the present invention is useful for controlling bacteria causing periodontal disease. The vaccines of the present invention can be used in the field of veterinary medicine, in particular for treating companion animals, and can also be used to maintain public health against the bacteria described herein known to cause periodontal disease.

Vaccines of the invention are useful for controlling bacteria that cause, spread, or act as vectors, for example, the diseases of humans and companion animals described herein. Vaccines of the present invention are particularly useful for controlling bacteria present in companion animals in that they can be administered using any known method of administration, including oral, parenteral, intranasal, subcutaneous or topical administration, and the like. .

In another aspect of the invention, there is provided a composition comprising a vaccine of the invention in combination with a compatible adjuvant, diluent or carrier. In a preferred embodiment, the vaccine formulation of the invention contains one or more bacterial and / or one or more subunit proteins of the invention and preferably consists of an aqueous suspension or solution in injectable form buffered at physiological pH. It is. In another preferred embodiment, the vaccine formulation of the invention comprises three or more porphyromonas species, for example blood. Whale B43, p. Salibosa B104 and Oh. It consists of an inactivated whole cell preparation of Dentikanis B106.

The invention also provides a method of treating or preventing a bacterial infection comprising treating with an effective amount of a vaccine or vaccine formulation of the invention. Reference to treatment should be understood to include prevention as well as alleviation of established signs of bacterial infection.

Vaccines and vaccine formulations of the present invention can be used to induce a response that prevents pathological alteration of the periodontal disease characteristics caused by periodontal disease-causing bacteria. In vaccine formulations, immunogenic amounts of bacteria, purified proteins, nucleic acids, or combinations thereof are preferably mixed with conventional vaccine adjuvants and physiological vehicles suitable for use in mammals.

Vaccine formulations for preventing periodontal disease in companion animals use one or more isolated and purified inactivated or attenuated bacteria, purified polypeptides (eg, native proteins, subunit proteins or polypeptides), one or more of these Can be prepared by mixing with a compatible adjuvant, diluent or carrier. Preferably, the polypeptide sequence is a subunit protein selected from the group comprising FimA (SEQ ID NOs: 103-110) and OprF (SEQ ID NOs: 120-128).

Examples of FimA and OprF fragments that can be used in diagnostic polypeptide or vaccine preparations include, but are not limited to, ACNKDNEAEPVV, YPVLVNFESNNYTYTGDAVEK, TGPGTNNPENPITESA, NDNNNKDFVDRLGA, DLNGQINRLRREVEELSKRPVSCPECPDV and ADPTGDTQYNERLSERRAKAV (SEQ ID NO: 129-134). Subunit proteins can be expressed recombinantly, alone or in fusion to other polypeptide sequences or proteins. Other polypeptide sequences or proteins may include, but are not limited to, for example, poly-his tags, MBP, thioredoxin or GST. The present invention also provides a polynucleotide sequence or gene encoding any of the aforementioned subunit proteins. The polynucleotide sequence of a bacterium is either fimA and oprF, or a fragment or variant thereof (this fragment or variant is directed to fimA and oprF At least about 90%, 95% or 99% homology), or complementary polynucleotide sequences that hybridize under high stringency conditions, or a combination thereof. Preferably, the polynucleotide sequence of the present invention may be used to an amino acid fragment which can be used to generate antibodies to the fimA or oprF DNA molecule, or FimA or OprF of the present invention amplify a fimA or oprF DNA molecule encoding.

For DNA-based therapies, vehicles can be used that can deliver or transport heterologous nucleic acids to host cells. Expression vehicles can include components that regulate targeting, expression, and transcription of nucleic acids in a cell-selective manner, as known in the art. Expression vehicles can include promoters that regulate the transcription of the heterologous material, which can be constitutive promoters or inducible promoters that allow selective transcription. Enhancers may optionally be included that may be required to obtain the required level of transcription.

The vector may be introduced into a cell or tissue by any of a variety of methods known in the art. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, MI (1995), Vegas et al., Gene Targeting, CRC Press, Ann Arbor, MI (1995) , [RL Rodriguez Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988)], for example, stable or transient transfection with recombinant viral vectors, lipofection, electrolysis Perforation and infection methods.

The invention also provides a combination of a vaccine having one or more inactivated or attenuated bacteria, nucleotide sequences or polypeptide sequences of the invention with one or more additional immunogenic components. Such combination vaccines may surprisingly have a much higher effect than expected by simply adding the effect of administering each component individually in the vaccinated animals. Thus, combination vaccines can promote synergistic production of antibodies in animals.

In a preferred embodiment, the combination vaccine of the present invention comprises at least three porphyromonas species, for example blood. Whale B43, p. Salibosa B104 and Oh. It consists of one or more additional bacterial or viral immunogenic components along with the inactivated whole cell preparation of Dentikanis B106. Additional immunogenic components suitable for use in the combination vaccines of the invention include canine measles virus (CDV), canine adenovirus-2 (CAV-2), canine parvovirus (CPV), canine parainfluenza virus (CPI) and Canine coronavirus (CCV), but is not limited thereto.

Vaccines of the invention may be prepared by combining one or more inactivated or attenuated bacteria, nucleotide sequences or polypeptide sequences of the invention with a pharmaceutically acceptable carrier, preferably an adjuvant.

Vaccine formulations suitable for the present invention include injectable liquid solutions or suspensions. Solid forms suitable for solution or suspension in a pharmaceutically acceptable liquid carrier may also be prepared before injection. Vaccine formulations may be emulsified. The active immunogenic component is preferably mixed with adjuvants that are pharmaceutically acceptable and compatible with the active immunogenic component. Suitable auxiliaries include mineral gels such as aluminum hydroxide; Surfactant materials such as lysolecithin; Glycosides such as saponin derivatives such as Quil A or GPI-0100 (US Pat. No. 5,977,081); Cationic surfactants such as DDA, pluronic polyhydric alcohols; Polyanions; Non-ionic blocking polymers such as Pluronic F-127 (B.A.S.F., USA); Peptides; Mineral oils such as Montanide ISA-50 (Seppic, Paris, France), Carbopol, Amphigen (Hydronics, Omaha, NE), Alhydrogel (Alhydrogel; Superfos Biosector, Federix Sund, Denmark); Oil emulsions such as mineral oils (such as Bayol F / Arlacel A) and emulsions of water, or emulsions of vegetable oils, water and emulsifiers (such as lecithin); Alum, cholesterol, rmLT, cytokines and combinations thereof, but is not limited thereto. The immunogenic component may also be incorporated into liposomes or conjugated to polysaccharides and / or other polymers used in vaccine formulations. Additional materials that may be included in the products used in the process of the invention include, but are not limited to, one or more preservatives such as ethylenediaminetetraacetic acid (EDTA), disodium salts or tetrasodium salts of merthiolates.

The subject to which the vaccine is administered is preferably a companion animal, most preferably a dog or a cat.

The vaccine of the invention is preferably present in a single dosage form in the vaccine formulation. For the purposes of the present invention, when administered, the immunogenic amount comprises about 1 × 10 ⁴ to 1 × 10 ¹³ inactivated bacterial cells, 0.1 μg to 1 mg purified protein, or 0.1 μg to 10 mg nucleic acid. do. In vaccine formulations containing multiple components, the same or less immunogenic amounts may be usefully employed.

Appropriate therapeutically effective dosages can be readily determined by one skilled in the art based on the immunogenic amount, treatment conditions and physiological characteristics of the animal. Accordingly, the vaccine formulation provides a dose of sterile formulation comprising an immunogenic amount of the active ingredient, wherein the active ingredient is one or more bacteria, proteins, nucleic acids, or any combination thereof. In the presence of additional active agents, these single doses can be easily adjusted by those skilled in the art.

Preferred dosage regimens include administering one or more dosages of the desired vaccine composition, wherein the antigen content of each fraction is as mentioned above. The effective dose (immunization) of the vaccine of the present invention can also be estimated by extrapolation from the dose-response curve obtained from the model test system. The method of administering the vaccine of the present invention can be any route suitable for delivering the vaccine to a host. Examples include oral, intradermal, intramuscular, intraperitoneal, subcutaneous, intranasal routes, and scarification (e.g., wounding the outermost layer of skin using a bifurcated needle) There is a route through, but is not limited thereto. However, preferably the vaccine is administered subcutaneously or by intramuscular injection. If desired, other methods of administration may be used, such as, for example, intradermal, intravenous, intranasal or tonsil routes.

In studies with each of the vaccine compositions described above, it was found that primary immunization of young animals (after 8 weeks of age) is initiated by booster doses administered preferably at 12 and 16 weeks of age. It is proposed to re-vaccinate each year.

The vaccines of the present invention are administered and administered according to good medical practice taking into account the clinical conditions of the individual subject, the site of administration and the method of administration, the dosing regimen, the subject age, sex, weight and other factors known to the medical practitioner.

The present invention also provides a kit for preventing periodontal disease in companion animals. In one embodiment, the kit provides a container comprising a therapeutically effective amount of a composition for preventing periodontal disease in a companion animal. Also provided are the same or different containers comprising a pharmaceutically acceptable carrier that can be used in the composition. The kit may additionally include an adjuvant that can be used to help produce a reaction to the composition of the present invention. The kit may also comprise a dispenser for dispensing the composition, preferably in a single dosage form. This dispenser may comprise, for example, a metal or plastic foil, such as a blister pack. The kit may be accompanied by a label or instructions describing administering the composition to prevent periodontal disease in a companion animal. In addition, a composition comprising the vaccine composition of the present invention formulated in a pharmaceutically acceptable carrier is prepared and placed in an appropriate container and indicated for the treatment of the indicated periodontal disease state.

Determination of the efficacy of the vaccine

A specific protective mechanism induced by the vaccines and vaccine compositions of the present invention is to induce antibodies and / or cellular immune responses in vaccinated animals, as shown in the in vivo animal test described below.

The bacteria, polynucleotides, polypeptides, vaccines and vaccine compositions of the invention may be useful for treating or preventing companion animal periodontal disease, bovine side disease, coronary heart disease (dog) or systemic infection (dog). In addition, the compositions of the present invention can be used in certain diseases of companion animals, such as coronary heart (or vascular or arterial) diseases, mumps, bad breath, gingivitis, periodontitis, stroke, atherosclerosis, hyperlipidemia, low weight fetus It may also be useful for treating or preventing increased preterm birth rates, bacterial vaginosis and delayed intrauterine development (IUGR).

In another aspect of the invention, a method of assessing the efficacy of a vaccine against one or more periodontal pathogenic bacteria in an animal is provided. The present invention shows that vaccines against one or more periodontal pathogenic bacteria can be evaluated in animal species such as mice or dogs, especially dogs.

According to the present invention, various periodontal pathogenic bacteria (Porphyromonas, Bacteroides, Prebotella, Tannerella) (Tenerella Forsythesis, formerly Bacteroides forcitus) and Treponema Vaccines designed to treat or prevent periodontal disease in humans or companion animals caused by) can be assessed using the methods described above. The vaccine may contain inactivated or attenuated bacteria, polypeptides or polynucleotides of any of the above bacterial species.

The efficacy of the vaccine can be assessed by introducing the challenge cultures into vaccinated and non-vaccinated animals separately and comparing the clinical signs of these two animals. The challenge culture may consist of the same periodontal bacteria as the vaccine. However, the challenge culture may contain bacteria other than those present in the vaccine to assess any cross protection the vaccine may have against other bacterial species.

In the present invention, it is preferable to introduce the challenge culture into the root canal of the animal teeth in which the prosthesis is placed after the root material is removed. Infection cultures typically range from about 1 × 10 ² to about 1 × 10 ¹² per challenge dose. Colony forming units (CFU); Preferably from 1 × 10 ⁵ to about 1 × 10 ¹¹ colony forming units (CFU) per challenge dose; Even more preferably from about 5 × 10 ⁷ to about 5 × 10 ¹⁰ per challenge dose. It contains colony forming units (CFU).

Clinical signs of the disease that can be evaluated include increased levels of one or more periodontal pathogenic bacteria, or changes in alveolar bone volume, especially in the periapical region of the alveolar bone, in gingival fluid, plaque, infected bone or gingival Change in the amount of alveolar bone in Bone change can be quantified, for example, by roentgen imaging measurements.

The invention is illustrated in more detail by the following non-limiting examples and the accompanying drawings.

Example  One

Companion animal Fissure  Sample

Microbes from dogs and cats examined in veterinary clinical trials for periodontal disease, or dogs examined at Pfizer Terre Haute or Pfizer Sandwich facilities for normal check-up Samples were taken. Dogs with periodontal pockets greater than 3 mm and cats with periodontal pockets greater than 2 mm were included in the study. Tooth index (gum index and periodontal index) and periodontal pocket depth were recorded. Individual respirator points (Henry Schein, Melville, NY) were inserted into the periodontal pocket aseptically. When removing it, the pre-reduced Anaerobically Sterile (PRAS) Anaerobic Dental Transport (ADT) medium (Unaero System; Morgan Hillis, Calif.), Where the blotter points were pre-reduced and sterilized under anaerobic conditions Immediately inserted into the vial containing.

The vial was transferred to a Bactron IV anaerobic chamber (Sheldon Manufacturing, Cornelius, OR) and treated under 90% N ₂ , 5% H ₂ , 5% CO ₂ . The blotter points were sterilely placed in 50 μl of PRAS brain heart mix (BHI) medium (Unaerobe system) and vortexed for 30 seconds. 1: 100 and 1: 1000 dilutions were prepared in BHI medium. 100 μl aliquots of 1: 100 and 1: 1000 dilutions were spread on PRAS Brucella Blood Agar (BRU) plates (unairbed system). Plates were incubated at 37 ° C. for 5-7 days in an anaerobic chamber. The total number of bacterial colonies and the number of Black Pigmented Anaerobic Bacteria (BPAB) colonies were counted. Individual BPAP colonies were transferred to new BRU plates and incubated again as above.

clinical Isolate  Specialization

For each clinical isolate a number of biochemical and 16S rRNA DNA sequencing were performed using primers D0056 and D0057 (SEQ ID NO: 1 and SEQ ID NO: 2) to determine the genus and species. Individual isolates were streaked on BRU plates. Kanamycin, vancomycin and colistin discs (unaerobic system) were placed on the agar surface to determine the KVC resistance pattern of each isolate. In addition, indole and catalase tests (unaerobic systems) were performed on purified colonies. Each isolate was transferred to an egg yolk agar (EYA) plate (unaerobic system) to determine lipase and recytase production patterns. This data is shown in Table 2.

Isolates were typed based on their 16S rRNA DNA sequences. Individual well isolated colonies were used as templates for polymerase chain reaction (PCR) amplification of 16S rRNA regions using triples of primers D0056 and D0057 (SEQ ID NO: 1 and SEQ ID NO: 2). PCR was performed using 1 × PCR buffer (Life Technologies; Rockville, MD), 1.0 mM MgCl ₂ , primers of 1.25 μM each, deoxy-NTP of 300 μM each, and 2.5 U platinum Pfx DNA polymerase. It was performed at 50 μl reaction volume containing (Life Technologies). The following PCR cycle conditions were used: denaturation step at 94 ° C. for 2 minutes; 30 cycles of denaturation at 94 ° C. for 40 seconds, annealing at 60 ° C. for 40 seconds, and elongation at 72 ° C. for 1 minute; Final elongation step at 72 ° C. for 2 minutes; And final cooling at 4 ° C. GeneAmp 9700 thermal cycler (Perkin Elmer Applied Biosystems; Foster City, CA) was used for all PCR amplifications.

The resulting PCR product was purified using a PCR purification kit (Promega Corp .; Madison, Wisconsin) and collected by isolation. The purified PCR product was then desalted by dropwise analysis on 25 ml sterile water using a 0.025 μm nitrocellulose filter (Millipore Corp .; Bedford, Mass.). Purified dechlorinated PCR products were subjected to ABI automated DNA sequence analyzer (University of Texas Genetics Core Facility, Houston, TX and Larks Technologies Inc., Houston, TX). DNA sequences were analyzed using dideoxy terminal reaction at. Double stranded DNA sequences were obtained using synthetic oligonucleotide primers D0056, D0057, PFZ175-AP1, PFZ175-AP2, and PFZ175-AP3 (SEQ ID NOS: 1-5; Table 1, respectively). Using the generated DNA sequences, the existing DNA databases were examined using the BLAST-N program obtained from The National Center for Biotechnology Information (USA).

Bacterial isolates were categorized based on the closest match identified by database survey. The B106 isolates did not exactly match, suggesting that it may represent a novel bacterial genus / species (see below). All isolates and their respective properties are listed in detail in Table 2. The top nine strains most frequently isolated are: Whale B43 (4 sample sandwiches), p. Kansi B46 (dog sample VHUP 1B), p. Circumdentaria B52 (cat sample VHUP 2A), p. Whale B69 (cat sample VHUP 3A), p. Sukhumentaria B97 (dog sample TH 1bC), p. Kanjinjivalis B98 (dog sample TH 1aC), p. Salibosa B104 (dog sample TH 1bC), o. Dentikanis B106 (dog sample TH 1bE), and p. Endodontalis B114 (dog sample TH 2bA).

The distribution of the isolates is shown in Table 3.

The isolates listed above are the most frequently identified and represent species present in very high proportions of dogs or cats.

The following animal periodontal isolates were deposited on August 9, 2001 in the American Type Culture Collection (ATCC, Manassas University Boulevard 10801, Virginia, USA 20110): P. Whale B43 (PTA-3618), p. Kansul B46 (PTA-3619), p. Sukhumentaria B52 (PTA-3620), p. Whale B69 (PTA-3621), p. Sukhumentaria B97 (PTA-3622), p. Kanjinjivalis B98 (PTA-3623), p. Salibosa B104 (PTA-3624), oh. Denticanis B106 (PTA-3625), and p. Endodontalis B114 (PTA-3626).

Identification of new genus / species (Odoribacter dentikanis)

The inventors have identified a number of clinical isolates in which the 16S rRNA sequence does not have a highly similar identity to existing databases, indicating that the bacteria are novel isolates. One group of these isolates was believed to represent new species. Based on the data presented herein, we describe this novel species Odoribacter Gen. A new trick called Odoribacter gen.nov . Odoribacter denticanis type strain is strain B106 ^T (= ATCC PTA-3625 ^T ; = CNCM I-3225 ^T ).

Odoribacter dentikanis B106 ^T was isolated from adult hybrid male dogs (do not know age) with periodontal disease. Paper dot samples were obtained from the mandibular first molar with a periodontal pocket depth of 4 mm and a gingival index of 1. Samples were processed as described above. Purified cells were treated with RapID ANA II Clinical Trial Kit (Remel; Renex, KS) and Vitek Anaerobic Biological Identification (ANI) Card System (BioMerieux Inc .; Durham, NC). Biochemical analysis using Briefly, bacterial cells on the cultured Brucella blood agar plates were resuspended in McFarland # 3 equivalents. The suspension was added to RapID ANA II test wells or Vitek ANI cards and incubated at 37 ° C. for 4 hours. The biochemical test results were recorded according to the incubation period.

Table 4 is oh. RapID ANA II test results for Dentikanis B106 ^T and six related bacteria are shown. Of the 18 tests performed with the RapID ANA II kit, only two (LGY and IND) are available. Positive for Denticanis B106 ^T. Comparison results: Porphyromonas jinjivalis ATCC 33277, Prebotel intermedia ATCC 25611, Tanerella forsythesis ATCC 43037, Bacteroides tetaotaomicron ATCC 29148, Bacteroides pragilis ATCC 25285, and Bacteroides splancinicus ATCC 29572 showed 5, 4, 10, 12, 9 and 8 positive tests, respectively.

^* The response element in each test: URE, urea; BLTS, p-nitrophenyl-β, D-disaccharide; αARA, p-nitrophenyl-α, L-arabinoside; ONPG, o-nitrophenyl-β, D-galactoside; αGLU, p-nitrophenyl-α, D-glucoside; βGLU, p-nitrophenyl-β, D-glucoside; αGAL, p-nitrophenyl-α, D-galacttoside; αFUC, p-nitrophenyl-α, L-fucoside; NAG, p-nitrophenyl-n-acetyl-α, D-glucosaminide; PO ₄ , p-nitrophenylphosphate; LGY, Louisyl-Glycine-β-naphthylamide; GLY, glycine-β-naphthylamide; PRO, proline-β-naphthylamide; PAL, phenylalanine-β-naphthylamide; ARG, arginine-β-naphthylamide; SER, serine-β-naphthylamide; PYR, pyrrolidoneyl-β-naphthylamide, and IND, tryptophan.

^† Abbreviation: P, positive; N, negative; V, variability.

Five. Dentikanis B106 ^T , p. Biochemical reaction patterns of Jinjivalis ATCC 53977, and Fusobacterium nucleatum subspecies Nucleatum ATCC 23726, were measured using a Vitek ANI card (Table 5). Five. Denticanis B106 ^T showed only two positive tests (INDOL and TTZ), whereas p. Jinji Balis ATCC 53977 showed five positive tests (INDOL, PO4, NAG, BANA, and TTZ). Similar results were found in all 12 tests common to RapID-ANA II and Vitek ANI cards. Using the database supplied by Vitek, o. Denticanis B106 ^T isolate was found to have a confidence level of 63% (not shown). It was found to be inconsistent with Nucleatum. This discrepancy may be due to the absence of livestock PAB isolates from the Vitek database.

^* The response element in each test: IND, tryptophan; PO ₄ , p-nitrophenyl phosphate; PHC, p-nitrophenyl phosphate choline; ONPG, o-nitrophenyl-β, D-galactopyranoside; AGAL, p-nitrophenyl-α, D-galactopyranoside; BGLU, p-nitrophenyl-β, D-glucopyranoside; AGLU, p-nitrophenyl-α, D-glucopyranoside; BGUR, p-nitrophenyl-β, D-glucuronide; BLAC, p-nitrophenyl-β, D-lactoside; AMAN, p-nitrophenyl-α, D-mannopyranoside; AFUC, p-nitrophenyl-α, L-fucopyranoside; BFUC, p-nitrophenyl-β, D-fucopyranoside; BXYL, p-nitrophenyl-β, D-xylpyranoside; AARA, p-nitrophenyl-α, L-arabinofuranoside; NAG, p-nitrophenyl-N-acetyl-glucosaminide; BANA, N-benzyl-DL-arginine p-nitroanilide; LEU, L-leucine p-nitroanilide; PRO, L-proline p-nitroanilide; ALA, L-alanine p-nitroanilide; LYS, L-lysine p-nitroanilide; GGT, gamma-glutamyl p-nitroanilide; TTZ, triphenyl tetrazolium; ADH, arginine; URE urea; GLU, glucose; TRE, trehalose; ARA, arabinose; RAF, raffinose; XLY, xylose.

^† Abbreviation: +, positive; -, voice.

Five. 16S rRNA genes from Dentikanis B106 ^T were PCR amplified in triplicate using primers D134 (SEQ ID NO: 169) and D57 (SEQ ID NO: 2). PCR products were combined, purified, desalted and directly DNA sequenced. 5) at the National Center for Biotechnology Information. BLAST-N of a non-overlapping nucleotide database using Dentikanis B106 ^T 16S rRNA gene sequence [Altschul et al. , 1990] Research. It has been shown that Dentikanis B106 ^T isolate is associated with members of the genus Bacteroides. Five. The dentikanis B106 ^T 16S rRNA gene sequence was most closely related to the 16S rRNA gene sequence of Bacteroidetes sp. Oral clone FX069 (Accession No. AY134906) showing 96.3% identity over 1,465 bp. Bacteroidetes spp. Oral clone FX069 was isolated from human patients suffering from NUP during a study to define bacterial species associated with necrotic ulcerative periodontitis (NUP) in HIV-positive patients [Paster, BJ, et al. , "Phylogeny of Bacteroides , Prevotella , and Porphyromonas spp. And related bacteria", J Bacteriol. (1994), 176: 725-732.

Phylogenetic analysis based on 16S rRNA gene sequence was performed using CLUSTAL X version 1.81 software [Thompsom, JD, et al. "CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice", Nucleic Acids Res. (1994) 22: 4673-4680. Phylogenetic trees were created using the neighbor-connection method [Saitou, N. & Nei, M. "The neighbor-joining method: a new method for reconstructing phylogenetic trees." Mol. Biol. Evol. (1987) 4: 406-425. Bootstrap values were obtained using 1000 replicates. 1 is oh. The results of the phylogenetic analysis for Dentikanis B106 ^T isolate are shown. Phylogenetic trees previously published for the cytotopa-flavobacter-bacteroides (CFB) group, with placement of major genera (Porphyromonas , Bacteroides, Prebotella, Tanerella, etc.) Is consistent with [Paster et al. , 1994; Sakamoto, M., et al. , "Reclassification of Bacteroides forsythus (Tanner et al . 1986) as Tannerella forsythensis corrig., Gen. Nov., Comb.nov." Int. J Syst. Evol. Microbiol. (2002) 52: 841-849; Shah, HN & Collins, D. "Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis , and Bacteroides endodontalis in a new genus, Porphyromonas." Int. J Syst. Bacteriol. (1998) 38: 128-131.

Five. Dentikanis B106 ^T is rain. Splancinicus [Werner, H., et al., "A new butyric acid-producing Bacteroides species: B. splanchnicus n. Sp." Zentralbl. Bakteriol. (1975) 231: 133-144, several oral isolates, and a large population of Bacteroides, including many non-cultured strains. Five. Dentikanis B106 ^T and B. The 71.0% bootstrap confidence value at the branching point of Splancinicus indicates a moderately high degree of certainty that these two organisms are involved. However, rain in the genus Bacteroides. There has been a historical discussion of the placement of Splancinicus [Paster et al. , 1994; Shah et al. , 1998]. Pasta is rainy. It was suggested that splancholicus could represent the new genus. Shah et al. It has been shown that Splancinicus shares many chemical classification properties with members of the genus Porphyromonas. The data of the present inventors is non- Splancinicus / B. This is consistent with the hypothesis because the Dentikanis B106 ^T group diverges closer to Porphyromonas than to the genus Bacteroides (FIG. 1).

Five. Approximately 580-bp region (Table 1) of 16S rRNA genes from 25 additional canine clinical isolates of dentikanis were PCR amplified using the D56 and D57 primers above. PCR product was purified and desalted. The DNA sequence of the PCR product was then determined. Five. Partial 16S rRNA sequences (SEQ ID NOS: 138-162) from dentikanis isolates were found clustered together in groups of identical or highly conserved sequences, where the largest estimate between these groups was 97% over the analyzed region. It was the same. 2 is oh. Showing the results of phylogenetic analysis of dentikanis isolate partial 16S rRNA gene sequence; Representative isolates from each group of relevant sequences were selected for analysis. Based on this observation, it can be concluded that all of these isolates are various strains of the same species.

Five. Clinical isolates of dentikanis were also obtained from cats (Table 1). The same approximately 580-bp region of the 16S rRNA gene was amplified from these isolates by PCR using D56 and D57 primers. DNA sequences (SEQ ID NOs: 163-168) obtained from the purified PCR product were aligned with sequences obtained from canine isolates. Sort result is feline o. Denticanis isolates represent various strains of the same species found in dogs.

Oh, for the genus Porphyromonas. To further analyze the relationship of Dentikanis B106 ^T , we PCR amplified the fimA gene (in duplicate ) using degenerate PCR primers D122 (SEQ ID NO: 84) and D123 (SEQ ID NO: 85) (Invitrogen Corp.). .)). PCR products were purified, desalted, combined and directly DNA sequenced. BLAST-P study of non-duplicate polypeptide databases using FimA amino acid sequences from the National Institute of Biological Technology Information [Altschul et al. , 1990]. It has been shown that Dentikanis B106 ^T FimA protein is associated with FimA protein from members of the genus Porphyromonas. 3 shows the phylogenetic relationship of various FimA protein sequences. Very strong relationship (100% bootstrap value). It is present between the dentikanis B106 ^T protein and some type I fimbrillin proteins of the Porphyromonas spp.

Five. Denticanis B106 ^T and Porphyromonas gulae B43 were subjected to scanning electron microscopy to measure cell morphology. Briefly, 36-hour liquid culture in modified PYG medium was centrifuged at 1800 × g. Replace the medium with 3% EM grade glutaraldehyde (Polysciences, Inc., Warrington, Pa.) In 0.1 M phosphate buffer, pH 7.3, and incubate at 4 ° C. for 1 hour. It was. The cells were then washed twice in 0.1 M phosphate buffer (pH 7.3) and suspended in buffered 1% osmium tetraoxide (Polysciences, Warrington, Pa.) At room temperature for 1 hour. Samples were dehydrated in a series of ethanol series and finally dehydrated by immersion in hexamethyldisilazane for 15 minutes and then air-dried for 1 hour. The sample was fixed on an aluminum stub and coated with 200 kPa of gold on a Polaron SEM autocoating unit (Polaron Intruments, Inc., Heartfield, PA). It was. Each sample was examined with an ISI, DS-130 scanning electron microscope (ISI, Inc., Tokyo, Japan). Images were captured with a Yellow (Noran) 4485 digital beam control interface (Noran Intruments, Inc., USA). FIG. 4 shows scanning electrons of P. Gulas B43 and O. Denticanis B106 ^T. FIG. The short rod-shaped form (average length 1.25 μm) of P. oyster B43 (FIG. 4A) is a typical member of the genus Porphyromonas, in contrast, O. denticanis B106 ^T has a tapered end and mean It was fusiform with cell length of 5.9 μm (FIG. 4B) O. Denticanis B106 ^T morphology was tanerella under these growth conditions. Similar to forsythesis (Tanner et al ., 1986). blood. Come on and oh. The morphological differences between denticanis are outside the genus Porphyromonas. Dentikanis Support the placement further.

Culture Conditions for Porphyromonas Species

Since standard growth media (Berce Leachation (BHI) and Chopped Meat Carbohydrate (CMC) media) for Porphyromonas species contain animal products that are not suitable for vaccine production, growth media that do not contain these components are required. . Various media compositions with and without the addition of hemin and vitamin K tested their ability to support growth equivalent to the growth of BHI or CMC. Both PYG-complete media and ME-complete media were at equivalent levels of BHI. Cave supported the growth of B43 (PTA-3618) (FIG. 5). PYG-complete media was removed due to its ability to obtain high density cultures during fermentation. Cholla B43 (PTA-3618) was selected as the growth medium. The medium contains the following components: 3% phyton (Becton Dickinson, Cockcaseville, MD), 0.3% yeast extract (Becton Dickinson), 0.3% glucose (St. Louis, MO) Sigma Corp.), 0.05% Sodium Thioglycolate (Becton Dickinson), 0.5% Sodium Chloride (Sigma Corporation), 5 μg / ml Hemin (Sigma Corporation) (added after autoclave), 0.5 μg / ml menadione (Sigma Corporation) (added after autoclave), and 0.2% sodium bicarbonate (Sigma Corporation), pH 7.0.

blood. Dolphin B43 (PTA-3618) is a Brucella blood at 37 ° C. under 90% N ₂ , 5% CO _{2 in a} Bactron IV anaerobic chamber (Shel Labs, Cornelius, Oregon, USA). Cultures were commonly cultured on agar plates (anaerobic systems) or in complete PYG medium or BHI for 3 to 5 days (plates) or 24 to 48 hours (liquid culture). In case of whole cell bacterial vaccine production, blood. Boula B43 (PTA-3618) was incubated in a BioFlo 3000 bioreactor using 5 liters of PYG complete medium. Culture medium in the vessel was made anaerobic by sparging 95-99.5% N ₂ and 0.5-5% CO ₂ immediately after the autoclave. 0.02% blood in reduced culture medium. Seeds were seeded with B43 (PTA-3618) stock, incubated at 37 rpm with a stirring speed of 100 rpm, and the pH was maintained at 7.0 by automatic addition of NaOH. During the cultivation, the container was sparged with both N ₂ and CO ₂ periodically. While bacterial cells were collected after 36 to 48 hours at an OD ₆₀₀ of 2.0 to 3.5, the cells were still logarithmic.

Pathogenicity Testing of Clinical Isolates in Mice

Nine isolates (P. goulash B43, P. kansulsi B46, P. circumdentaria B52, P. gulsa B69, P. circumdaria B97, P. kanzinjivalis B98, P. salibosa B104, o Denticanis B106, and P. endodonthalis B114) was tested in a mouse periodontal bone loss model for its pathogenicity. Three-week-old male-matched male Balb / c CyJ mice (Jackson Laboratories, Bar Harbor, Maine, USA) with a weight estimation of 14-15 g were used for this study. This animal was housed in a barrier cage unit at positive pressure, and food pellets and water standard to this species were given randomly throughout the experiment. The pallet used was particle Bed O'Cobbs to minimize impact on gum tissue. After receipt, all animals were acclimated for 5-7 days. To lower the competing oral flora, the animals were placed in a mixture of sulfamatoxazole and trimetapririm for 10 days (10 ml drinking water; approximately 2 mg and 0.4 mg / ml respectively) followed by a 5-day washout period. Serum samples were obtained from each mouse tail vein bleeding. Animals were infected by Gabbaz with 0.5 ml of a suspension of the appropriate bacterial strain of approximately 1 × 10 ¹⁰ cfu / ml in 1% carboxymethylcellulose. Additional drops were given to the oral cavity. This infection was repeated at least 2 of 3 times (Monday, Wednesday, and Friday).

Experiment 1 was defined as Tuesday after the first infection. All animals were slaughtered on day 2. Post-infection serum was collected as a microbial sample. The jaw flesh of each mouse was removed, stained and scored for horizontal bone loss under a microscope. Scoring was repeated three times to reduce operator error. Mean bone loss is expressed as mean bone loss / site / chin (mm). Statistical analysis of the obtained data was carried out with Systat (version 9), SigmaStat (version 2), and SigmaPlot (version 2000) (SPSS Science Inc.) (Illinois, USA). Available from Chicago, Ltd.). Table 6 shows the numerical results for the top nine isolates.

Summary of Mouse Periodontal Disease Pathogenicity Trial. Isolate Mouse count Bacteria Source Average bone loss (mm) Standard Deviation SEM Siamese 32 N / A 0.0843 0.0118 0.00211 blood. Earnest Ballis 53977 16 human 0.106 0.0139 0.00347 blood. Jinji Balis W50 16 human 0.0948 0.0116 0.0029 blood. Earl Bally's B40 A 16 dog 0.106 0.0138 0.00357 blood. Earl Bally B40 B 16 dog 0.115 0.0114 0.00284 blood. Whale B43 16 dog 0.112 0.0163 0.00407 blood. Kansul B46 16 dog 0.101 0.014 0.00362 blood. Circumdentaria B52 16 cat 0.0924 0.00836 0.00209 blood. Whale B69 16 cat 0.114 0.0129 0.00322 blood. Circumdentaria B97 16 dog 0.0855 0.0143 0.00368 blood. Kanjinjivalis B98 16 dog 0.111 0.0136 0.0034 blood. Salibosa B104 16 dog 0.102 0.0107 0.00286 Five. Denticanis B106 16 dog 0.124 0.0167 0.00417 blood. Endodontalis B114 16 dog 0.0994 0.0223 0.00557

Each of these showed statistically significant bone loss in this model.

6 graphically illustrates net bone loss. Mean alveolar bone level (white enamel border-alveolar ridge) was obtained in mm at 14 maxillary sites and the mean value for each jaw was measured. For each experimental group, the mean value for each jaw was summed and divided by the total number of animals in that group to obtain a group mean.

7 graphically shows a comparison of net bone loss. Mean alveolar bone level (white enamel border-alveolar ridge) was obtained in mm at 14 maxillary sites and the mean value for each jaw was measured. For each experimental group, the mean value for each jaw was summed and divided by the total number of animals in that group to obtain a group mean. The net bone loss was measured by subtracting the mean value of Siamese infection from each experimental group. Positive control set to 100% 53977). blood. Genjivalis W50 is an incomplete fimbrinated strain with reduced virulence in this animal model.

These data indicate that the following clinical isolates can produce high levels of bone loss in mouse models of periodontal disease. Whale B43 (PTA-3618), p. Whale B69 (PTA-3621), p. Kanjinjivalis B98 (PTA-3623) and o. Denticanis B106 (PTA-3625). The following clinical isolates induced moderate bone loss in the mouse periodontal model: blood. Kansul B46 (PTA-3619), p. Salibosa B104 (PTA-3624), and p. Endodontalis B114 (PTA-3626). The following clinical isolates caused minimal bone loss in the mouse periodontal model: blood. Cercumdenaria B52 (PTA-3620) and p. Sukhumentaria B97 (PTA-3622). While varying amounts of bone loss have been observed between clinical isolates, it should be noted that the amount of bone loss observed in each case is far greater than that observed in Siamese infected mice. Based on these data, it can be estimated that the top nine clinical isolates can cause periodontal disease alone or in cooperation with other bacteria.

Production of bacterial cells and genomic DNA

Bacterial species were incubated anaerobicly for 48 hours at 37 ° C. in BHI or complete PYG. Cells from 1-3 ml of culture were pelleted by centrifugation, washed once with an equivalent amount of anaerobic PBS, recentrifuged, and resuspended with 1/10 volume of anaerobic PBS.

Genomic DNA was purified from 5 ml of culture of bacterial species cultured anaerobicly for 48 hours at 37 ° C. in BHI or complete PYG. Wizard Genomic DNA Extraction Kit (Promega Corp.) was used for all genomic DNA preparation.

Clinical From isolate  Cilia fimbrial A) gene Cloning

The fimA gene was topped using a combination of PCR primers D0067 (forward; SEQ ID NO: 6), D0078 (forward; SEQ ID NO: 8), D0097 (forward; SEQ ID NO: 9), D0068 (reverse; SEQ ID NO: 7) and D0098 (reverse; SEQ ID NO: 10). PCR amplification from genomic DNA isolated from 10 clinical isolates. PCR was performed using 1 × PCR buffer (Life Technologies), 1.0 mM MgCl ₂ , 1.25 μM each primer, 300 M each deoxy-NTP, and 2.5 U platinum Pfx DNA polymerase (Life Technologies). It was carried out in μl reaction volume. The following PCR cycle conditions were used: 2-minute denaturation step at 94 ° C .; 30 cycles of denaturation at 94 ° C. for 40 seconds, annealing at 60 ° C. for 40 seconds, and elongation at 72 ° C. for 1.5 minutes; Final elongation step at 72 ° C. for 5 minutes; And final cooling to 4 ° C. GeneAmp 9700 thermal cycler (Perkin Elmer Applied Biosystems, Foster City, Calif.) Was used for all PCR amplifications. The amplified product was visualized on a 1.2% E-gel (Invitrogen, Carlsbad, CA).

PCR products were A-tailed with 10 units of KlenTaq polymerase (Ab Peptides, Inc., St. Louis, MO) for 5 minutes at 72 ° C. The resulting product was immediately cloned into the pCR2.1-TOPO vector (Invitrogen) using the manufacturer's protocol and cloned into E. coli. E. coli Top10F '(Novagen, Madison, WI) was transformed. Transformants with recombinant plasmids with correct insert DNA were subjected to DNA sequencing using colony PCR, restriction enzyme cleavage, and DyeDeoxy termination reactions on ABI automated DNA sequences (Lark Technologies, Inc.). ) Was confirmed. Synthetic oligonucleotide primers (SEQ ID NOs: 6, 7, 8, 11-42) were used to obtain double stranded DNA sequences.

Blood into the expression plasmid. Whale B43 Fima  Cloning of genes

For the purpose of high level protein expression, avoid blood. The native B43 (PTA-3618) fimA gene was cloned into pBAD / HisA expression vector (Invitrogen). Blood in BHI in which genomic DNA was anaerobicly incubated at 37 ° C. for 2 days using a genomic DNA extraction kit (Promega Corporation). Purified from 5 ml of culture of B43. Three primers of the f imA gene were PCR amplified using primers D0097 and D0098 (SEQ ID NO: 9 and SEQ ID NO: 10). PCR was performed using 1x PCR buffer (Life Technologies), p. 50 μl reaction volume containing 50 ng of B43 genomic DNA, 1.0 mM MgCL ₂ , 1.25 μM each primer, 300 μM each deoxy-NTP, and 2.5 U platinum Pfx DNA polymerase (Life Technologies).

The following PCR cycle conditions were used: 2-minute denaturation step at 94 ° C .; 5 cycles of denaturation at 94 ° C. for 40 seconds, annealing at 58 ° C. for 40 seconds, and elongation at 72 ° C. for 1.5 minutes; 30 cycles of denaturation at 94 ° C. for 40 seconds, annealing at 65 ° C. for 40 seconds, and elongation at 72 ° C. for 1.5 minutes; Final elongation step at 72 ° C. for 5 minutes; And final cooling to 4 ° C. GeneAmp 9700 thermal cycler (Perkin Elmer Applied Biosystems) was used for all PCR amplifications. PCR products were purified with a PCR flap kit (Promega Corporation). Purified PCR product and pBAD / HisA were double digested with Hin dIII and Xho I at 37 ° C. for 3 hours. In the middle of the cleavage, 5 units of shrimp alkaline phosphatase (SAP) (Amersham Pharmacia Biotech, Inc., Fitzkataway, NJ) were added to the vector cleavage. The cleaved DNA was purified with a DNA Clean-Up Kit (Promega Corporation). Purified Hin dIII / Xho I cleaved PCR product was treated with Hin dIII / Xho I cleaved SAP with p4D4 / Xho I cleaved SAP for 18 hours at 16 ° C. in the presence of 1 × T4 DNA ligase buffer. Ligation into HisA. A portion of the resulting ligation mixture is water soluble. Transformed into E. coli Top10F 'cells (Novagen). The recombinant plasmid pBAD: B43fimA4 containing the fimA gene in the correct orientation was found. The resulting recombinant FimA contains the terminal vector-coding sequence (MGGSHHHHHHGMASMTGGQMGRDLYDDDDKDRWGSELEICSQYHMGI, SEQ ID NO: 135), followed by the maturation site of FimA starting at asparagine-20. These plasmids were purified for further protein expression analysis. Transformed into E. coli BL21 cells (Novagen).

Expression and Purification of Recombinant FimA Proteins

this. Coli Freeze working stock of BL21 / pBAD: B43fimA4 and thaw and seed at 1: 5000 dilution into Luria broth containing 100 μg / ml ampicillin (1% tryptone, 0.5% yeast extract, 0.5% NaCl) And grow at 100 rpm agitation rate at 37 ° C. in a 5-liter working volume BioFlo 3000 bioreactor (New Brunswick Scientific, Edison, NJ) until A ₆₂₅ is 2.5 v 3.5 I was. L-arabinose was then added to the culture at 0.1% final concentration to induce FimA expression. Cultures were incubated for an additional 3 hours. Expression of recombinant FimA was detected by SDS-PAGE and Western blot analysis using anti-Express serum (Invitrogen) (FIG. 8). Recombinant FimA protein had a predicted molecular weight of 45 kDa.

E. coli expressing recombinant FimA from 5 liter fermentation. Coli Wet cells of BL21 transformants were harvested by centrifugation and resuspended in phosphate-buffered saline. Cells were lysed mechanically. After centrifugation, the pellets were removed. The supernatant was passed over a Ni ²⁺ -affinity column and eluted with an imidazole gradient. Fractions containing recombinant protein were combined, dialyzed to remove imidazole, and filter-sterilized with a 0.2 μm filter.

From clinical isolates oprF Cloning of genes

blood. Gene PG32 (Genbank Accession No. AF175714), Oligonucleotide Primer D0086 (SEQ ID NO: 43), D0087 (SEQ ID NO: 44), and KWK-Pg-03 (SEQ ID NO: 172) based on the sequence of the Genjivalis strain W50 oprF homologue 45) was designed and synthesized (Life Technologies). For PCR, Primer D0086 (SEQ ID NO: 43) was run on D0087 (SEQ ID NO: 44) or KWK-Pg-03 (SEQ ID NO: 45), 200 μM each dNTP, 7.5 U in 50 μl final sample volume of 1 × PC2 buffer (Ab peptides). Used with Klen Taq 1 (Ab peptides) and 0.15 U cloned Pfu (Stratagene, La Jolla, CA) thermostable polymerase. The reaction is blood. Whale B43, p. Kansul B46, p. Sukhumvitaria B52, p. Whale B69, blood. Sukhumvitaria B97, p. Kanjinjivalis B98, p. Salibosa B104, oh. Denticanis B106, and p. This was done in triplicates using cell suspensions washed with templates from Endodontalis B114 or purified genomic DNA. Amplification was carried out as follows: denaturation (94 ° C., 9 min); 30-40 cycles of denaturation (94 ° C., 30 seconds), annealing (55-60 ° C., 30 seconds), and polymerization (72 ° C., 1.5 minutes); Then final elongation at 72 ° C. for 7 minutes.

blood. Primer KWK-Ps-04b (SEQ ID NO: 81) was used with KWK-Ps-06b (SEQ ID NO: 83) for polymerase chain amplification of the oprF homolog from Kanjinjivalis B98. blood. Salibosa For amplification of homologs from B104, primer KWK-Ps-04b (SEQ ID NO: 81) was used with KWK-Ps-05b (SEQ ID NO: 82). Five. Dentikanis For amplification of the gene from B106, primer KWK-Ps-02 (SEQ ID NO: 79) was used with KWK-Ps-03 (SEQ ID NO: 80). The reaction is strain S. Kanjinjivalis B98, p. Salibosa B104, and o. Dentikanis Three runs were performed using chromosomal DNA purified as a template from B106. Amplification was carried out as follows: denaturation (94 ° C., 9 min); 30-35 cycles of denaturation (94 ° C., 30 seconds), annealing (61-72 ° C., 30 seconds), and polymerization (72 ° C., 1.5 minutes); Then final elongation at 72 ° C. for 7 minutes.

PCR amplified gene products were visualized by separating on 1.0% agar gel (Sigma). PCR products were purified with QIAquick ™ PCR Purification Kit (Qiagen, Valencia, Calif.) And each set of three samples collected. These fragments were then sequenced directly to block the introduction of sequence artifacts by mutations that occur during PCR amplification and subsequent cloning steps. The combined mixtures were then subjected to direct DNA sequencing (Lark Technologies) using a DyeDeoxy termination reaction on an ABI automated DNA sequencer. Synthetic oligonucleotide primers (SEQ ID NOs: 46-75) were used to sequence all of the DNA strands of the amplified product.

blood. Whale B43, p. Kansul B46, p. Sukhumvitaria B52, p. Whale B69, blood. Sukhumvitaria B97, p. Kanjinjivalis B98, p. Salibosa B104, oh. Denticanis B106, p. Kanjinjivalis B98, p. Salibosa B104, oh. Dentikanis B106, and p. The nucleotide sequence encoding the OprF homolog from P. endontalis B114 is shown in SEQ ID NOs: 111-119. Sequences corresponding to the 5 'and 3' primers used for PCR amplification of each gene were removed because they may not represent the actual sequence of the gene in each of the representative strains. ORFs encoding SEQ ID NOs: 111-119 are shown in SEQ ID NOs: 120-128, respectively. In each case of the encoded ORF, the amino terminal sequence still retained the properties of the prokaryotic single sequence even when coding for the 5 'primer [von Heijne, 1985, J. Mol. Biol. 184: 99-105; Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G., 1997 Protein Engineering, 10: 1-6]. Each ORF was subjected to a BLAST (Basis Local Align Search Tool) program [Altschul, SF, Gish, W., Miller, W., Myers, EW, Lipman, DJ, 1990, J. Mol. Biol. 215: 403-410] for the nucleotide and protein databases present. Avoid headings that share maximal homology. It was PG32 from Jinji Bally.

Blood into the expression plasmid. Whale B43 oprF Cloning of genes

For the purpose of recombinant protein expression, the gene encoding OprF was cloned with the sequence encoding its signal peptide. OprF was cloned with oligonucleotide primers KWK-Pg-06 (SEQ ID NO: 76) and KWK-Pg-03 (SEQ ID NO: 45). Amplified from B43. For polymerase chain amplification, 2 sets of 50 μl containing chromosomal DNA as template, 1 × PC2 buffer, 200 μM each dNTP, 50 pMol each primer, 7.5 U Klen Taq 1 and 0.15 U cloned Pfu thermostable polymerase The reaction was set up. Amplification was carried out as follows: denaturation (94 ° C., 9 min); 30 cycles of denaturation (94 ° C., 30 seconds), annealing (60 ° C., 30 seconds), and polymerization (72 ° C., 1.5 minutes); Then final elongation at 72 ° C. for 7 minutes. After amplification, samples were purified (QIAquick ™ PCR Purification Kit) and collected. Purified PCR products were cloned directly into the TA cloning site of both pBAD-TOPO and pBAD / Thio-TOPO (Invitrogen). Ligand product was Max Efficiency e. Transformed into E. coli DH5α cells. The predicted amino terminal sequence of the coding protein expressed from pBAD-TOPO: OprF consists of starting with the vector-coding sequence MGSGSGDDDDKLALM (SEQ ID NO: 136), immediately following the sequence (SEQ ID NO: 120) at glutamine-13 of OprF. Clones containing the appropriate plasmids were identified and purified plasmids were isolated from small broth cultures with a QIAprep Spin Miniprep kit (Qiagen). This plasmid. Transformed into E. coli BL21 cells (Novagen) and clones containing the appropriate plasmids were identified.

The predicted amino terminal sequence of the coding fusion protein expressed from pBAD / Thio-TOPO: oprF should consist of the thioredoxin protein and the 14 amino acid residue linker, immediately following the sequence (SEQ ID NO: 120) at glutamine-13 of OprF. . Clones containing the appropriate plasmids were identified and purified plasmids were isolated from small broth cultures with QIAprep spin miniprep kits. This plasmid. Transformed into E. coli BL21 cells and clones containing the appropriate plasmids were identified.

The oprF gene, which lacked the signal peptide coding sequence, was also cloned into two different λ expression plasmids. These plasmids all encode a temperature-sensitive λ refresher cI 857 that inhibits expression from the λ promoter at 30 ° C. At 42 ° C., the repressor is inactivated and expression from the λ promoter is enabled, resulting in high levels of transcription and copying. For cloning into these vectors, oprF was cloned into oligonucleotide primers KWK-Pgu-14 (SEQ ID NO: 77) and KWK-Pgu-15 (SEQ ID NO: 78). Amplified from B43. Washed blood as template for polymerase chain amplification. Two 50 μl reactions were set up with Dola B43 cells, 1 × PC2 buffer, 200 μM each dNTP, 50 pMol each primer, 7.5 U Klen Taq 1 and 0.15 U cloned Pfu thermostable polymerase. Amplification was carried out as follows: denaturation (94 ° C., 9 min); 45 cycles of denaturation (94 ° C., 30 seconds), annealing (55 ° C., 30 seconds), and polymerization (72 ° C., 1.5 minutes); Then final elongation at 72 ° C. for 7 minutes. After amplification, samples were collected, digested with restriction enzymes, and an excess was produced using the same enzyme and also compatible with the linearized plasmid. After restriction cleavage, PCR fragments and plasmids were purified (QIAquick ™ PCR Purification Kit; Qiagen Corporation), ligated to E. coli. Transformed into E. coli DH5α cells (Novagen). The predicted amino terminus consisted of the vector-coding sequence MGTTTTTTSLHM (SEQ ID NO: 137), immediately following the sequence (SEQ ID NO: 120) at glutamine-13 of OprF. The protein expressed from the second plasmid will consist only of Vector-encoding Met, followed by glutamine-13 (SEQ ID NO: 120) of OprF. Clones containing the appropriate plasmids were identified and the plasmids were isolated from small broth cultures with the QIAprep spin miniprep kit (Qiagen Corporation). These plasmids were e.g. Transformed into E. coli BL21 cells and individual clones containing the appropriate plasmids were identified.

Recombination OprF  Expression and Purification of Proteins

E. expressing recombinant Oprf (fused to SEQ ID NO: 137 at the N-terminus). E. coli BL21 cells were used for expression studies. Freeze stocks were thawed and seeded at 1: 5000 dilution in 2 × YT medium (1.6% tryptone, 1% yeast extract, 0.5 NaCl) containing 50 μg / ml kanamycin sulfate, with A ₆₂₅ between 2.5 and 3.5 Were grown in a 5-liter running volume BioFlo 3000 bioreactor (New Brunswick Scientific; Edison, NJ) at agitation rate 100 rpm at 29 ° C. The culture was then transferred to 42 ° C. to induce Oprf expression. Cultures were incubated for an additional 3 hours. Aliquots were collected at various time points, centrifuged and resuspended in reducing sample buffer. All samples were analyzed on 10% NuPAGE gels (Invitrogen, USA) (FIG. 9).

 E. expressing recombinant Oprf from 5 liter fermentation. Wet cells of E. coli BL21 transformants were harvested by centrifugation and resuspended in phosphate buffered saline. Cells were lysed mechanically. After centrifugation, the pellets were removed. The supernatant was passed through an ion exchange column and eluted using a NaCl gradient. Fractions containing the recombinant protein were pooled, dialyzed to remove NaCl, and filter-sterilized using a 0.2 μm filter.

Whole cell Bacterin  Formulation

blood. A 5 liter batch of gullet B43 was grown in a fermentor as described above and split into 1 liter portions. Cells in each 1 liter fraction (total 4.4 × 10 ¹² cells blood. Whale B43 cells) exposed to 0.4% formalin for 24 hours at 23 ° C., 10 mM binary ethylene-imine (BEI) at pH 8.5 for 48 hours at 37 ° C., heated to 60 ° C. for 30 minutes per day for two consecutive days. And by treatment exposed to air for 48 hours. After BEI treatment, the activity of BEI was removed by treatment with 50 mM sodium thiosulfate. Cells were collected by centrifugation. The resulting cell pellet was resuspended in 220 ml PBS to give a final concentration of 2 × 10 ¹⁰ cells / ml. 7 ml of each inactivated cell was mixed with 7 ml of MPL + TDM adjuvant (Sigma Corp.) to obtain a final concentration of 1.0 × 10 ¹⁰ cells / ml.

Eight other top clinical isolates (P. Kansulsi B46, P. Sukhumvitaria B52, P. Gulsa B69, P. Sukhumvitaria B97, P. Cangingivalis B98, P. Salibosa B104, O. Den) Tikanis B 106, and P. endodontalis B114) or other whole cell bacterin preparations of colored anaerobic bacteria can be prepared in the same manner.

Homologous Vaccine Efficacy in Mice

In homologous vaccine efficacy studies, the above-mentioned inactivated blood in MPL + TDM adjuvant at 3 week intervals. Mice were immunized by injecting 0.2 ml of each BW cell twice. Two weeks after further booster immunization, the mice were evacuated as described above. Infected with B43 cells. After 42 days of infection, mice were killed and treated as described above. Table 7 shows the numerical results of the bone loss measurements.

Mouse Homeopathic Vaccine Efficiency Findings group Vaccinogen Challenge Average bone loss Standard Deviation SEM Net loss % Bone loss (a) % Bone loss (b) A PBS with RIBI MPL + TDM Adjuvant Never 0.0686 0.00862 0.00216 0 NA (c) NA B PBS with RIBI MPL + TDM Adjuvant Pg 53977 0.112 0.0107 0.00266 0.0434 100 NA C PBS with RIBI MPL + TDM Adjuvant Pg B43 0.093 0.0188 0.00471 0.0244 NA 100 D Formalin deactivation blood containing Freund's adjuvant. Earnest Ballis 53977 Pg 53977 0.098 0.0146 0.00364 0.0294 67.7 NA E Formalin deactivation blood containing RIBI MPL + TDM adjuvant. Earnest Ballis 53977 Pg 53977 0.0932 0.0109 0.00271 0.0246 56.7 NA F Formalin deactivation blood containing RIBI MPL + TDM adjuvant. Whale B43 Pg B43 0.082 0.0128 0.00319 0.0134 NA 54.9 G Formalin deactivation blood containing RIBI MPL + TDM adjuvant. Whale B43 Pg B43 0.107 0.0151 0.0039 0.0384 NA 157.4 H Heat inactivation with RIBI MPL + TDM adjuvant p. Whale B43 Pg B43 0.0845 0.0113 0.00281 0.0159 NA 65.2 I Air inactivation with RIBI MPL + TDM adjuvant p. Whale B43 Pg B43 0.0746 0.00691 0.00173 0.006 NA 24.6

(a) Percent calculated based on group B as positive control

(b) Percent calculated based on group C as positive control

(c) NA = not applicable

10, 11, and 12 show these results graphically. 11 shows the bone loss rate of the control experiment. Formalin-activated blood. Vaccines containing Genjivalis 53977 and Freund's complete / incomplete adjuvant or MPL + TDM adjuvant should be avoided. Bone loss caused by infection with Genjivalis 53977 was reduced by about 32% and 43%, respectively. 12 shows bone loss rate for test group experiments. Formalin-, heat-, or air-inactivated blood. Vaccine containing B43 and MPL + TDM adjuvant should be avoided. Bone loss caused by infection with Whale B43 was reduced by about 45%, 35%, and 75%, respectively. Based on the data, formalin-, air- or heat-activated blood. It can be concluded that the native B43 vaccine is effective in reducing bone loss induced in the ultra-infected model. Extrapolating the data to clinical settings, the three vaccines may prove effective in the prevention of periodontal disease and in the treatment of periodontal disease.

Heterologous Vaccine Efficacy Study in Mice

In heterologous vaccine efficacy studies, formalin-activated blood in MPL + TDM adjuvant at three week intervals. Whale B43 or formalin-activated blood. Salibosa B104 and Oh. Mice were immunized by injecting 0.2 ml of each of Dentikanis B106 cells twice. After 2 weeks of additional immunization, avoid mice as described above. Whale B43, p. Whale B69, blood. Salibosa B104 or Oh. Infected with Dentikanis B106 cells. After 42 days of infection, mice were killed and treated as described above. Table 8 shows the numerical results of the bone loss measurements.

Mouse heterologous vaccine efficacy findings group Vaccinogen Inactivation method Test infection Average bone loss Standard Deviation SEM Net goal loss % Bone loss ^a % Bone loss ^b % Bone loss ^c % Bone loss ^d A PBS NA Never 0.088 0.0112 0.00299 0 0 0 0 0 B PBS NA blood. Whale B43 0.101 0.0103 0.00266 0.013 100 NA ^e NA NA C PBS NA blood. Whale B69 0.115 0.0112 0.00289 0.027 NA 100 NA NA D PBS NA blood. Salibosa B104 0.101 0.0132 0.00352 0.013 NA NA 100 NA E PBS NA Five. Denticanis B106 0.0994 0.0135 0.0035 0.0114 NA NA NA 100 F blood. Whale B43 formalin blood. Whale B43 0.0901 0.016 0.00412 0.0021 16.15 NA NA NA G blood. Whale B43 formalin blood. Whale B69 0.104 0.0166 0.00443 0.016 NA 59.26 NA NA H blood. Whale B43 formalin blood. Salibosa B104 0.0926 0.0119 0.00319 0.0046 NA NA 35.28 NA I blood. Whale B43 formalin Five. Denticanis B106 0.102 0.0124 0.00333 0.014 NA NA NA 122.8 J blood. Salibosa B104 / O. Denticanis B106 formalin blood. Whale B69 0.102 0.0124 0.00333 0.014 NA 51.85 NA NA

Avoid ^a bone loss rate. Calculated for Whale B43 infected mice.

^b Avoid bone loss. Calculated for Cavage B69 infected mice.

^c Avoid bone loss. Calculated for Salibosa B104 infected mice.

^d Avoid bone loss. Calculated for Dentikanis B106 infected mice.

^e NA, not applicable

13, 14, 15, 16 and 17 graphically show these results. 9 shows net bone loss for the experiment. 14 is blood. Bone loss rate for the B43 infected group. Formalin-activated blood. Do not bleed with B43 and MPL + TDM supplements. Bone loss caused by infection with Whale B43 was reduced by about 84%. 15 is blood. Bone loss rate for the B69 infected group. Formalin-activated blood containing MPL + TDM adjuvant. Whale B43 and formalin-activated blood. Salibosa B 104 / O. Dentikanis B106 vaccine is blood. Bone loss caused by infection with Whale B69 was reduced by about 40% and 49%, respectively. 16 is p. Bone loss rate for Salibosa B 104 infected group. Formalin-activated blood. Do not bleed with B43 and MPL + TDM supplements. Bone loss caused by Salibosa B 104 was reduced by about 65%. 17 is o. Bone loss rate for Dentikanis B106 infected group is shown. Formalin-inactivated blood containing MPL + TDM adjuvant. Whale B43 vaccine oh. Cross protection against challenge with Dentikanis B106 failed. Based on the data, formalin-inactivated blood supplemented with MPL + TDM. Do not let B43 bleed as well as homologous challenge. It can be concluded that it can protect against heterologous challenge by whale B69. Extrapolating the data to clinical settings, it can be demonstrated that multivalent vaccines are effective in the prevention of periodontal disease and in the treatment of periodontal disease.

Recombination Fima  And Oprf  Serological Studies in Mice

In subunit vaccine serology studies, blood purified by recombination in QuilA / cholesterol adjuvant at three week intervals. Whale B43 FimA or recombinantly expressed and purified blood. Mice were immunized with two injections of 0.2 ml of B43 OprF each. Mice were killed before first vaccination and two weeks after further immunization. Table 9 shows the numerical results and FIGS. 18 and 19 show the results graphically.

Mouse Subunit Vaccine Serological Studies rFimA ELISA rOprF ELISA group Vaccinogen Before vaccination After vaccination Before vaccination After vaccination A Brine 50 50 50 50 B rFimA + QAC 50 138889 NA NA C rOprF + QAC NA NA 50 118

Example  2

In dog challenge models Periodontal Pathogenicity

Ten-year-old beagle dogs with adult dentition were used in this study. Anesthetize the animal and access the root canal of the mandibular premolar and the first molar using a Midwest Dental Products Corp (Des Plaines, IL) in a Schein Ultima 2000 dental unit with a high speed handpiece. It was. Access to the root canal was confirmed by passing a veterinary barbed broach (21 mm, size # 5; Roydent Dental Products; Johson City, TN) into the root canal to a depth close to the root canal depth. Repeated passage of the barbed broach removes connective tissue, tubular and neural tissue. Major bleeding did not occur well; Hemostasis was performed with sterile paper points (Henry Schein Inc.) placed in the root canal. At this time, it is important to remove any accidental contamination of the root canal during perforation and emptying of the root canal. Thus, the root canal was flushed with 10% bleach solution. In order to create a suitable surface for the placement of the prosthesis, 40% sulfate gel (Scotchbond Etching Gel, 3M; St. Paul, MN) was used to etch the enamel surrounding the access ports. In order to prevent any effect of residual bleach solution or acid gel on the viability of the challenge material, an angioplasty needle (27 ga., Densply Pharmaceutical; York, PA) and a large amount of sterile saline are used to retract the root canal Flushed. The access area and root canal were dried with sterile paper points.

Infectious material was collected on Brucella blood agar (Anaerobe Systems, Morgan Hill, CA). The whale strain B69 was grown and prepared by incubation at 37 ° C. under an anaerobic environment (5% H ₂ , 5% CO ₂ , 90% N ₂ ). Cells were harvested and resuspended in sterile SSYG medium. An challenge dose of about 7.5 × 10 ⁸ colony forming units (CFUs) was then introduced into the exposed root canal cavity of the selected tooth using an aneurysm needle. Five animals were administered challenge material (T01) and the other five animals were administered sterile SSYG medium (T02). The access port was then sealed with a combination of glass ionomer and photocurable dental composite prosthesis (Revolution 3: 3M).

Periodontal pathogenicity of the challenged organism was determined based on the change in density of the pubic bone surrounding the tooth. This assessment is made by measuring the pixel intensity of the digital radiograph obtained using Schick Compputed Dental Radiography (CDR ^® ) sensors and software at Weeks 0, 3, 6, 9, and 12 of the study week. lost. Radiographs of 6 to 8 teeth were obtained from each animal. To obtain baseline values, radiographs were taken just before the procedure. After challenge, radiographs were taken every 3 weeks for 12 weeks post- challenge. The radiographs were analyzed by two different methods. The first method consisted of subjective evaluation of the radiograph by a professional veterinarian in the analysis of dental radiographs. Briefly, this consisted of a trained observer examining the radiograph, noting the ideal, and marking the degree of abnormality for each dog on the Visual Analog Scale. Second, areas of infected bone apparent in the radiographs taken at 0, 6 and 12 weeks post challenge were measured using an area measurement tool in the radiograph software. This consisted of determining the approximate diameter of the visible lesion on the radiograph using a linear measurement tool in the Chic CDR software. From the distance of the result, the approximate area of the lesion was measured. For each dog these areas were summed and tabulated.

Analysis of radiographs using both methods suggested that the T01 group lost more periodontal bone than the T02 group. This study concludes that a possible challenge model has been developed. However, due to the difficulty of quantifying the changes occurring in the three-dimensional region (periodontal bone) based on two-dimensional measurements (radiography), an improved quantitation method will be required.

Example  3

Evaluation of Trivalent Vaccine Efficacy in Canine Infection Models

Following model development, studies were conducted to test the efficacy of trivalent vaccine formulations in dogs. Trivalent bacterins are formalin-activated blood. Whale (B43), p. Salibosa (B104), and o. Denticanis (B106) was contained and supplemented with 50 μg of Quil A and cholesterol for each dose. Each bacterin strain was collected at a concentration of approximately 1 × 10 ¹⁰ CFU / vaccine doses. Three groups of eight animals with adult teeth were used in this study. Dogs of the first group (T01) were vaccinated intramuscular (1M) with 1 ml of trivalent vaccine. The second group (T02) and the third group (T03) were falsely vaccinated with 1 ml of sterile saline. All dogs were administered intramuscularly (1M) three times with three weeks between doses. Three weeks after the final administration, the root material was removed and then the challenge material was introduced into the root canal of the mandibular premolar and the first molar. Heterologous blood, prepared as described in Example 1, in dogs T01 and T02. Whale strain (B69) 1 × 10 ¹⁰ CFU was vaccinated. For the purpose of measuring the effectiveness of the procedure, dogs in group T03 were challenged with sterile medium.

At 3, 6, 9, and 12 weeks post challenge, radiographs were obtained using a Helidont dental radiograph machine, a Chic ^® CDR (computer digital radiograph) capture system, and standard techniques. For this study, once a digital radiograph was obtained, the sequential images from each dog were first viewed using ImageJ (v1.28, NH: Bethesda, NH) using plug-in TurboReg ^® with scale rotation technique. Registration with each other via MD). The registered image set was corrected by third order polynomial using an external gray scale. The area of the bone surrounding the treated tooth, then excluding the tooth and air, was outlined and the average density of that area was recorded for each image. Thus, a numerical value representing the “whiteness” of the bone surrounding the tooth muscle could be objectively derived and used for analysis. This value is referred to as the “bone responsiveness score” and refers to the average bone density.

The results of the study are shown in FIG. Mean bone scores for animals in group T02 were reduced or whiter by 12 weeks after challenge when restored to pre-challenge levels. The average bone reactivity scores of groups T01 and T02 were also initially reduced and then returned to near normal levels by 12 weeks post challenge when they diverged. The procedural control of group T03 became whiter while the vaccinated animals of group T01 maintained mean values similar to pre-infection levels. Thus, dogs given the vaccine were able to recover bone density more quickly than dogs not vaccinated, despite an aneurysm challenge. In addition, analysis of serum collected at each observation point showed that the vaccinated animals developed high antibody titers against the vaccine strain and increased antibody cross-reactivity with the challenge strain (data suggested).

The radiograph becomes whiter rather than darker after challenge and becomes more recognizable in case of bone infection. The bone responds in a dual way to inflammation and infection, both cases increasing the surrounding bone which will lose the bone matrix and "block" the spreading infection. Mixed soluble-curable lesions are typically observed in several bone infections, but are affected by 1) toxicity of pathogens, 2) age of the animal, 3) heredity of the animal, and 4) the degree of trauma associated with stimulating inflammation. The animals used in this study are young (10-14 months of age) and have significant trauma to the teeth and surrounding bones involved in the challenge procedure. These factors may explain the reason for the increase in the density of the surrounding bones when considered in conjunction with what is known about how bones respond to infection and inflammation.

Example  4

Vaccine efficacy in canine challenge models

This vaccine efficacy study was similar to the manner described in Example 3. Each treatment group included 10 animals. Dogs of group T01 were vaccinated and challenged; T02 group was falsely vaccinated prior to challenge; T03 group was subjected to false positive vaccination and false positive challenge. Each vaccine contained formalin-inactivated blood at a concentration of 1 × 10 ¹⁰ CFU of each strain per dose. Whale strain B43, p. Salibosa strain B104 and o. It contained Dentikanis strain B106. The vaccination schedule was the same as described in Example 3. The challenge vaccine is blood. Alaska strain B69 and was administered 3 weeks after the third vaccination. The radiographs were obtained again at 3 week intervals but only up to 9 weeks post challenge and analyzed in a similar manner as described in Example 2. 22 shows the bone reactivity analysis results.

In this study, animals responded to challenge procedures with increased average bone reactivity scores. This means that the radiograph becomes darker in response to a decrease in bone density (FIG. 23). Radiographs showed significant lesions in both T01 and T02 groups, but the bones of unvaccinated animals were much less dense than the bones of vaccinated animals. At week 9, the mean bone reactivity scores of the vaccinated dogs in the T02 group were significantly different from the vaccinated group T01 (p = 0.05). The dogs in this study were evenly older than the dog of Example 2, and the operator's skills improved significantly. These factors are believed to contribute to a more typical response in connection with bone infection, namely a decrease in bone density. Thus, the anhydro-dental model of canine periodontitis is a novel and useful model for periodontitis research. This is valuable in vaccination / challenge studies related to vaccine assessment and can be used for antimicrobial and topical treatment studies.

Example  5

Evaluation of Systemic and Local Responsiveness to Vaccines

Evaluation of systemic and local tissue responses to trivalent vaccines in combination with other vaccines administered to dogs. At the beginning of the study, two two groups of about 6 weeks of age were vaccinated with 1M at 6, 9, and 12 weeks of age. Blood samples were collected before each vaccination. All dogs were examined daily for systemic and local responses one week after each vaccination. The blood temperature was measured and recorded on that date. Group T01 was vaccinated with trivalent bacterin (prepared as described in Examples 3 and 4) at a total antigen 3 × 10 ⁸ CFU concentration and supplemented with 50 μg of Quil A and cholesterol, respectively, per dose modified And 4 virus components: Canine Distemper Virus (CDV), Canine Adenovirus-2 (CAV-2), Canine Parvovirus (CPV), Canine Parainfluenza Virus CPI) or canine coronavirus (CCV) in combination. Group T02 was administered the same combination of trivalent bacterin and viral components at a concentration of 3 × 10 ⁸ CFU. The results in FIG. 24 showed that the trivalent vaccine did not result in a negative systemic or local response in the test group when added to the combination of viral components typically administered to puppies.

Throughout this application, various patents and scientific literature, including US patents, are cited by author and year and patent number. The specification of these documents and patents is incorporated herein by reference in their entirety in order to further detail the state of the art to which this invention belongs.

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Claims (15)

Companion animals including Porphyromonas gulae B43, Porphyromonas salivosa B104 and Odoribacter denticanis B106 inactivated whole cell preparations and pharmaceutically acceptable carriers A vaccine for treating or preventing periodontal disease in companion animals. The vaccine of claim 1, wherein the bacteria are inactivated by formalin. According to claim 1, Canine Distemper Virus (CDV), Canine Adenovirus-2 (CAV-2), Canine Parvovirus (CPV), Canine Parainfluenza virus (Canine Parainfluenza) A vaccine further comprising at least one of Virus (CPI) or Canine Coronavirus (CCV). A method of treating or preventing periodontal disease in a companion animal, comprising administering a vaccine according to any one of claims 1 to 3 to a companion animal in need of treatment or prevention of periodontal disease. To treat and prevent periodontal disease in a companion animal comprising a pharmaceutically acceptable carrier and inactivated whole-cell preparations of Porphyromonas salvia B104, Porphyromonas salibosa B104, and Odoribacter denticanis B106 in one or more containers. A kit comprising a composition, the kit further comprising a set of printed instructions indicating that the kit is useful for treating or preventing periodontal disease in a companion animal. delete delete delete delete delete delete delete delete delete delete

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